Isolated and purified 12R-lipoxygenase polypeptide and polynucleic acid encoding the same

ABSTRACT

Isolated and purified lipoxygenase proteins and nucleic acids are described. Particularly, a novel human 12R-lipoxygenase (12R-LO) protein and cDNA are described. Recombinant host cells, recombinant nucleic acids and recombinant proteins are also described, along with methods of producing each. Isolated and purified antibodies to 12R-LO, and methods of producing the same, are also described.

GRANT STATEMENT

[0001] This work was supported by NIH grant GM-53638. Humankeratinocytes were provided by the Tissue Core Laboratory of theVanderbilt Skin Disease Research Center, which is supported by grant5P30 AR41943-03 from the NIH/NIAMS. The U.S. Government has certainrights in the invention.

TECHNICAL FIELD

[0002] The present invention relates generally to isolated and purifiedlipoxygenase proteins and nucleic acids. More particularly, the presentinvention relates to an isolated and purified 12R-lipoxygenase and anisolated and purified polynucleic acid encoding the same.

[0003] The publications and other materials used herein to illuminatethe background of the invention, and in particular cases, to provideadditional details respecting the practice, are incorporated herein byreference, and for convenience, are referenced by author and date in thefollowing text, and respectively group in the appended list ofreferences. Table of Abbreviations 12R-LO 12R-lipoxygenase 12R-HETE12R-hydroxyeicosatetraenoic acid BSA Bovine serum albumin GC-MS Gaschromatography-Mass spectroscopy HAT Cell culture media comprisinghypoxanthine, aminopterin, and thymidine HETE Hydroxyeicosatetraenoicacid HPETE Hydroperoxyeicosatetraenoic acid H(P)ETE EitherHydroxyeicosatetraenoic acid or Hydroperoxyeicosatetraenoic acid HODEHydroxyoctadecadienoic acid HPLC High pressure liquid chromatography KLHKeyhole limpet hemocyanin PCR Polymerase chain reaction PFBPentafluorobenzyl ester PMA Phorbol-12-myristate-13-acetate RACE Rapidamplification of cDNA ends

BACKGROUND ART

[0004] The lipoxygenases are a structurally related family of non-hemeiron dioxygenases that function in the production of fatty acidhydroperoxides. Four lipoxygenases have been identified and cloned inhumans. Funk, C. D. (1993) Prog. Nuc. Acid Res. Mol Biol. 45:67-98;Matsumoto et al. (1988) Proc. Natl. Acad. Sci. USA 85: 26-30; Dixon etal. (1988) Proc. Natl. Acad. Sci. USA 85: 416-420; Funk et al. (1990)Proc. Natl. Acad. Sci. USA 87: 5638-5642; Izumi et al. (1990) Proc.Natl. Acad. Sci. USA 87:7477-7481; Yoshimoto et al. (1990) Biochem.Biophys. Res. Comm. 172:1230-1235; Sigal et al. (1988) Biochem. Biophys.Res. Comm. 157:457-464; Brash et al. (1997) Proc. Natl. Acad Sci. USA94:6148-6152). They oxygenate arachidonic acid in different positionsalong the carbon chain and form the corresponding 5S-, 12S- or15S-hydroperoxides (hydro(pero)xyeicosatetraenoic acids, H(P)ETEs).Three of these enzymes are known mainly from the blood cell types inwhich they are strongly expressed—the 55-lipoxygenase of leukocytes, the12S-lipoxygenase of platelets, and the 15S-lipoxygenase ofreticulocytes, eosinophils and macrophages. While these are the mostwidely recognized cellular sources, selective expression is documentedin other tissues. For example, both the 12S- and 15S-lipoxygenases aredetected in skin. Nugteren et al. (1987) Biochim. Biophys. Acta921:135-141; Henneicke-von Zepelin et al. (1991) J. Invest. Dermatol.97:291-297; Takahashi et al. (1993) J. Biol. Chem. 268:16443-16448;Hussain et al. (1994) Amer. J. Physiol. 266:C243-C253. The fourth of theknown human lipoxygenases, a second type of 15S-lipoxygenase, was clonedfrom skin and this enzyme is also expressed in prostate, lung, andcornea. Brash et al. (1997) Proc. Natl. Acad Sci. USA 94:6148-6152.

[0005] Interest in the biosynthesis of hydroxy derivatives ofarachidonic acid in skin stems from the role of essential fatty acidsand their derivatives in the structural integrity of normal epidermis(Burr et al. (1929) J. Biol. Chem. 82:345-367; Nugteren et al. (1985)Biochim. Biophys. Acta 834, 429-436; Nugteren et al. (1987) Biochim.Biophys. Acta 921:135-141), and from the potential involvement ofarachidonate metabolites in inflammatory and proliferative skin diseases(Hammarstrom et al. (1975) Proc. Natl. Acad. Sci. USA 72:5130-5134;Hussain et al. (1994) Am. J. Physiol. 266:C243-C253; Ziboh, V. A. (1996)Lipids 31:S249-S253). The major products of arachidonic acid metabolismin normal human skin or keratinocytes are 12-hydroxy- and15-hydroxyeicosatetraenoic acids (12-HETE and 15-HETE) (Nugteren et al.(1987) Biochim. Biophys. Acta 921:135-141; Hammarström et al. (1975)Proc. Natl. Acad. Sci. USA 72:5130-5134; Hussain et al. (1994) Am. J.Physiol. 266:C243-C253; Ziboh, V. A. (1996) Lipids 31: S249-S253;Burrall et al. (1988) J. Invest. Dermatol. 4:294-297; Green, F. A.(1989) J. Invest. Dermatol. 93:486-491; Holtzman et al. (1989) J. Clin.Invest. 84:1446-1453; Henneicke-von Zepelin et al. (1991) J. Invest.Dermatol. 97:291-297; Takahashi et al. (1993) J. Biol. Chem.268:16443-16448).

[0006] Biosynthesis of the 15-HETE is better understood in terms of theenzymes involved. It is formed almost exclusively as the 15S enantiomer(Baer et al. (1991) J. Lipid Research 32:341-347; Baer et al. (1993) J.Lipid Research 34:1505-1514.) and its production can be accounted for bythe 15S-lipoxygenases present in skin. Nugteren et al. (1987) Biochim.Biophys. Acta 921:135-141; Burrall et al. (1988) J. Invest. Dermatol.4:294-297; Green, F. A. (1989) J. Invest. Dermatol. 93:486-491;Henneicke-von Zepelin et al. (1991) J. Invest. Dermatol. 97:291-297;Takahashi et al. (1993) J. Biol. Chem. 268:16443-16448; Baer et al.(1991) J. Lipid Research 32:341-347; Baer et al. (1993) J. LipidResearch 34:1505-1514; Zhao et al. (1995) J. Lipid Res. 36:24444-2449;Brash et al. (1997) Proc. Natl. Acad Sci. USA 94:6148-6152.

[0007] Formation of the 12-HETE in human skin is more complex, in thatboth 12R and 12S enantiomers are produced (Holtzman et al. (1989) J.Clin. Invest. 84:1446-1453; Henneicke-von Zepelin et al. (1991) J.Invest. Dermatol. 97:291-297; Baer et al. (1991) J. Lipid Research32:341-347; Baer et al. (1993) J. Lipid Research 34:1505-1514. This isnot mainly attributable to autoxidation as the proportions of 12R and12S vary considerably and, aside from the 15S-HETE, comparable amountsof the other HETE regioisomers are not formed under the usual conditionsof in vitro biosynthesis. Formation of the 12S-hydroxy enantiomer can beaccounted for by the platelet-type of 12S-lipoxygenase that is aconstituent of normal and inflammatory human skin (Hussain et al. (1994)Am. J. Physiol. 266:C243-C253; Takahashi et al. (1993) J. Biol. Chem.268:16443-16448; Zhao et al. (1995) J. Lipid Res. 36:24444-2449; Brashet al. (1997) Proc. Natl. Acad Sci. USA 94:6148-6152). The enzyme orenzymes involved in the production of the-12R-enantiomer remainuncharacterized.

[0008] The first report of 12-HETE in human skin came in 1975, whenHammarström et al reported that the involved areas of epidermis inpsoriasis have markedly increased concentrations of free arachidonicacid and 12-HETE (Hammarström et al. (1975) Proc. Natl. Acad. Sci. USA72:5130-5134). Chiral analysis of the 12-HETE in psoriasis revealed thatthe major enantiomer is 12R-HETE (Woollard, P. M. (1986) Biochem.Biophys. Res. Commun. 136:169-175). It was shown subsequently that12R-HETE is a prominent product in other non-psoriatic proliferativedermatoses (Baer et al. (1995) J. Invest. Dermatol. 104:251-255), and itis also formed in normal human skin as the minor 12-HETE enantiomer(Holtzman et al. (1989) J. Clin. Invest. 84:1446-1453; Baer et al.(1993) J. Lipid Research 34:1505-1514).

[0009] It has been questioned whether the enzyme responsible for the12R-HETE synthesis is a cytochrome P450 or a lipoxygenase. TheP450-catalyzed synthesis of 12R-HETE is precedented in rat and humanliver microsomes and by purified cytochromes P450, as described inCapdevila et al. (1986) Biochem. Biophys. Res. Commun. 141:1007-1011;Oliw, E. H. (1993) Biochim. Biophys. Acta 1166:258-263; and Bylund etal. (1998) J. Pharmacol. Exp. Ther. 284:51-60. These well-defined P450reactions are, however, associated with the formation of many additionalproducts that are not typically formed in incubations of skin. Thealternative pathway, via a 12R-lipoxygenase, is precedented in a marineinvertebrate (Hawkins et al. (1987) J. Biol. Chem. 262:7629-7634;Hawkins et al. (1989) FEBS Lett. 247:9-12), but no R-lipoxygenase isknown in mammals.

[0010] Therefore, what is needed, then, is further characterization of12-lipoxygenase enzymes in vertebrates, particularly in mammals, andmore particularly in humans. A novel isolated and purified12R-lipoxygenase and a polynucleic acid encoding the same would havebroad utility due to its role in arachidonic acid metabolism, a criticalmetabolic pathway.

DISCLOSURE OF THE INVENTION

[0011] The present invention contemplates an isolated and purifiedvertebrate lipoxygenase polypeptide which metabolizes arachidonic acidto 12R-hydroxyeicosatetraenoic acid (12R-HETE). More preferably, apolypeptide of the invention is a recombinant polypeptide. Even morepreferably, a polypeptide of the present invention comprises a mammalian12R-lipoxygenase (12R-LO). Even more preferably, a polypeptide of thepresent invention comprises a human 12R-LO. Even more preferably, apolypeptide of the present invention comprises the amino acid residuesequence of SEQ ID NO:2.

[0012] The present invention also provides an isolated and purifiedpolynucleotide that encodes a lipoxygenase polypeptide which metabolizesarachidonic acid to 12R-HETE. In a preferred embodiment, apolynucleotide of the present invention comprises a DNA molecule from avertebrate species. A preferred vertebrate is a mammal. A preferredmammal is a human. More preferably, a polynucleotide of the presentinvention encodes a polypeptide designated 12R-LO. Even more preferred,a polynucleotide of the present invention encodes a polypeptidecomprising the amino acid residue sequence of SEQ ID NO:2. Mostpreferably, an isolated and purified polynucleotide of the inventioncomprises the nucleotide base sequence of SEQ ID NO:1.

[0013] In an alternative embodiment, the present invention provides anexpression vector comprising a polynucleotide that encodes a vertebratelipoxygenase polypeptide which metabolizes arachidonic acid to 12R-HETE.Also preferably, an expression vector of the present invention comprisesa polynucleotide that encodes human 12R-LO. More preferably, anexpression vector of the present invention comprises a polynucleotidethat encodes a polypeptide comprising the amino acid residue sequence ofSEQ ID NO:2. More preferably, an expression vector of the presentinvention comprises a polynucleotide comprising the nucleotide basesequence of SEQ ID NO:1. Even more preferably, an expression vector ofthe invention comprises a polynucleotide operatively linked to anenhancer-promoter. More preferably still, an expression vector of theinvention comprises a polynucleotide operatively linked to a prokaryoticpromoter. Alternatively, an expression vector of the present inventioncomprises a polynucleotide operatively linked to an enhancer-promoterthat is a eukaryotic promoter, and the expression vector furthercomprises a polyadenylation signal that is positioned 3′ of thecarboxy-terminal amino acid and within a transcriptional unit of theencoded polypeptide.

[0014] In yet another embodiment, the present invention provides arecombinant host cell transfected with a polynucleotide that encodes avertebrate lipoxygenase polypeptide which metabolizes arachidonic acidto 12R-HETE. SEQ ID NO:1 and SEQ ID NO: 2 set forth nucleotide and aminoacid sequences from an exemplary vertebrate, human. Also contemplated bythe present invention are homologous or biologically equivalentpolynucleotides and lipoxygenase polypeptides found in othervertebrates. Preferably, a recombinant host cell of the presentinvention is transfected with the polynucleotide that encodes human12R-LO. More preferably, a recombinant host cell of the presentinvention is transfected with the polynucleotide sequence of SEQ IDNO:1. Even more preferably, a host cell of the invention is a eukaryotichost cell. Still more preferably, a recombinant host cell of the presentinvention is a vertebrate cell. Preferably, a recombinant host cell ofthe invention is a mammalian cell.

[0015] In another aspect, a recombinant host cell of the presentinvention is a prokaryotic host cell. Preferably, a recombinant hostcell of the invention is a bacterial cell, preferably a strain ofEscherichia coli. More preferably, a recombinant host cell comprises apolynucleotide under the transcriptional control of regulatory signalsfunctional in the recombinant host cell, wherein the regulatory signalsappropriately control expression of the lipoxygenase polypeptide in amanner to enable all necessary transcriptional and post-transcriptionalmodification.

[0016] In yet another embodiment, the present invention contemplates aprocess of preparing a lipoxygenase polypeptide comprising transfectinga cell with polynucleotide that encodes a vertebrate lipoxygenasepolypeptide which metabolizes arachidonic acid to 12R-HETE to produce atransformed host cell; and maintaining the transformed host cell underbiological conditions sufficient for expression of the polypeptide. Morepreferably, the transformed host cell is a eukaryotic cell. Morepreferably still, the eukaryotic cell is a vertebrate cell.Alternatively, the host cell is a prokaryotic cell. More preferably, theprokaryotic cell is a bacterial cell of the DH5α strain of Escherichiacoli. Even more preferably, a polynucleotide transfected into thetransformed cell comprises the nucleotide base sequence of SEQ ID NO:1.SEQ ID NO:1 and SEQ ID NO:2 set forth nucleotide and amino acidsequences for an exemplary vertebrate, human. Also contemplated by thepresent invention are homologues or biologically equivalent lipoxygenasepolynucleotides and polypeptides found in other vertebrates.

[0017] In still another embodiment, the present invention provides anantibody immunoreactive with a vertebrate lipoxygenase polypeptide whichmetabolizes arachidonic acid to 12R-HETE. SEQ ID NO:1 and SEQ ID NO:2set forth nucleotide and amino acid sequences from an exemplaryvertebrate, human. Also contemplated by the present invention areantibodies immunoreactive with homologues or biologically equivalentlipoxygenase polynucleotides and polypeptides found in othervertebrates. Preferably, an antibody of the invention is a monoclonalantibody. More preferably, the lipoxygenase polypeptide comprises human12R-LO. Even more preferably, the lipoxygenase polypeptide comprises theamino acid residue sequence of SEQ ID NO:2.

[0018] In another aspect, the present invention contemplates a processof producing an antibody immunoreactive with a vertebrate lipoxygenasepolypeptide which metabolizes arachidonic acid to 12R-HETE, the processcomprising the steps of (a) transfecting a recombinant host cell with apolynucleotide that encodes a vertebrate lipoxygenase polypeptide whichmetabolizes arachidonic acid to 12R-HETE; (b) culturing the host cellunder conditions sufficient for expression of the polypeptide; (c)recovering the polypeptide; and (d) preparing the antibody to thepolypeptide. SEQ ID NO:1 and SEQ ID NO:2 set forth nucleotide and aminoacid sequences from an exemplary vertebrate, human. Preferably, the hostcell is transfected with the polynucleotide of SEQ ID NO:1. Even morepreferably, the present invention provides an antibody preparedaccording to the process described above. Also contemplated by thepresent invention is the use of homologues or biologically equivalentpolynucleotides and polypeptides found in other vertebrates to produceantibodies.

[0019] Alternatively, the present invention provides a process ofdetecting a vertebrate lipoxygenase polypeptide which metabolizesarachidonic acid to 12R-HETE, wherein the process comprisesimmunoreacting the polypeptide with an antibody prepared according tothe process described above to form an antibody-polypeptide conjugate,and detecting the conjugate.

[0020] In yet another embodiment, the present invention contemplates aprocess of detecting a messenger RNA transcript that encodes avertebrate lipoxygenase polypeptide which metabolizes arachidonic acidto 12R-HETE, wherein the process comprises hybridizing the messenger RNAtranscript with a polynucleotide sequence that encodes that polypeptideto form a duplex; and detecting the duplex. Alternatively, the presentinvention provides a process of detecting a DNA molecule that encodes avertebrate lipoxygenase polypeptide which metabolizes arachidonic acidto 12R-HETE, wherein the process comprises hybridizing DNA moleculeswith a polynucleotide that encodes a vertebrate lipoxygenase polypeptidewhich metabolizes arachidonic acid to 12R-HETE to form a duplex; anddetecting the duplex.

[0021] In another aspect, the present invention contemplates adiagnostic assay kit for detecting the presence of a lipoxygenasepolypeptide in a biological sample, where the kit comprises a firstcontainer containing a first antibody capable of immunoreacting with avertebrate lipoxygenase polypeptide which metabolizes arachidonic acidto 12R-HETE, with the first antibody present in an amount sufficient toperform at least one assay. Preferably, an assay kit of the inventionfurther comprises a second container containing a second antibody thatimmunoreacts with the first antibody. More preferably, the antibodiesused in an assay kit of the present invention are monoclonal antibodies.Even more preferably, the first antibody is affixed to a solid support.More preferably still, the first and second antibodies comprise anindicator, and, preferably, the indicator is a radioactive label or anenzyme.

[0022] In an alternative aspect, the present invention provides adiagnostic assay kit for detecting the presence, in biological samples,of a lipoxygenase polypeptide, the kits comprising a first containerthat contains a second polynucleotide identical or complementary to asegment of at least 10 contiguous nucleotide bases of a polynucleotidethat encodes a vertebrate lipoxygenase polypeptide which metabolizesarachidonic acid to 12R-HETE. Preferably, the polynucleotide encodeshuman 12R-LO.

[0023] In another embodiment, the present invention contemplates adiagnostic assay kit for detecting the presence, in a biological sample,of an antibody immunoreactive with a lipoxygenase polypeptide, the kitcomprising a first container containing a vertebrate lipoxygenasepolypeptide which metabolizes arachidonic acid to12R-hydroxyeicosatetraenoic acid that immunoreacts with the antibody,with the polypeptide present in an amount sufficient to perform at leastone assay. Preferably, the polypeptide comprises human 12R-LO.

[0024] Thus, a key aspect of this invention pertains to the discovery ofa novel 12R-lipoxygenase (12R-LO) protein and nucleic acid encoding the12R-LO protein. Preferred nucleic acid and amino acid sequences for12R-LO are described in SEQ ID NO:1 and SEQ ID NO:2.

[0025] It is another aspect of this invention that the novel 12R-LOprotein acts in the metabolism of arachidonic acid to12R-hydroxyeicosatetraenoic acid.

[0026] The foregoing aspects and embodiments have broad utility giventhe biological significance of the arachidonic acid pathway, as is knownin the art. By way of example, the foregoing aspects and embodiments areuseful in the preparation of screening assays and assay kits that areused to identify compounds that affect arachidonic acid metabolism, orthat are used to detect the presence of the proteins and nucleic acidsof this invention in biological samples. Additionally, it is well knownthat isolated and purified polypeptides have utility as feed additivesfor livestock and further polynucleotides encoding the polypeptides arethus useful in producing the polypeptides.

[0027] Following long-standing patent law convention, the terms “a” and“an” mean “one or more” when used in this application, including theclaims.

[0028] Some of the aspects and objects of the invention having beenstated hereinabove, other aspects and objects will become evident as thedescription proceeds, when taken in connection with the accompanyingdrawings as best described hereinbelow.

BRIEF DESCRIPTION OF THE DRAWINGS

[0029]FIG. 1 depicts analysis of deuterated 12-HETE formed in psoriaticscales.

[0030]FIG. 1A depicts chiral HPLC of the deuterium-labeled 12-HETE fromincubation of deuterated arachidonic acid in psoriatic scales. The12-HETE was chromatographed as the PFB ester on a Chiralcel OD columnusing a solvent of hexane:isopropanol (100:5, v/v) and detected by UVmonitoring at 235 nm.

[0031]FIG. 1B depicts partial mass spectra of the 12R-HETE from panel A(top), and 15S-HETE prepared from the same batch of deuteratedarachidonic acid using the soybean lipoxygenase (as described below).The samples were analyzed by negative ion/chemical ionization GC-MS asthe PFB ester trimethylsilyl ether derivatives by repetitive scanning inthe range m/z 390-404. The partial mass spectra are the average of allscans collected during elution of the peaks from the GC. Unlabeled12R-HETE is also detected in the psoriatic sample at m/z 391.

[0032]FIG. 2 sets forth cDNA (SEQ ID NO:1) and deduced amino acid (SEQID NO:2) sequences of the 12R-lipoxygenase of the present invention. Twoactively expressing clones of the new cDNA were sequenced and wereidentical. Putative iron ligands are boxed. The extra 31 amino acidsreferred to hereinbelow are underlined. The cDNA sequence, including 5′and 3′ UTR data, is set forth in the GenBank™/EMBL Data Bank withaccession number AF038461.

[0033]FIG. 3 depicts expression in Hela cells and identification of the12R-HETE product.

[0034]FIG. 3A depicts product analysis by normal-phase HPLC using anAlltech 5μ Econosil silica column (25×0.46 cm), a solvent system ofhexane:isopropanol:glacial acetic acid (100:1:0.1, by volume, changed tothe proportions 100:3:0.1 at 45 min), and a flow rate of 1.1 ml/min withon-line detection of raidiolabeled products using a Packard Flo-OneRadiomatic detector. Retention times of unlabeled HETE standards(co-injected with the ¹⁴C sample) are indicated on the chromatogram.

[0035]FIG. 3B depicts chiral analysis of the methyl ester derivative ofthe 12-HETE using a Chiralcel OD column with a solvent ofhexane:isopropanol (100:2, v/v) and a flow rate of 1.1 ml/min.

[0036]FIG. 4 depicts expression of the mRNA of the 12R-lipoxygenase ofthe present invention.

[0037]FIG. 4A is an autoradiograph of northern blot analysis of humankeratinocytes.

[0038]FIG. 4B is a photograph of agarose gel chromatography depictingdetection of the transcript of the 12R-lipoxygenase of the presentinvention in human keratinocytes and psoriatic scales by RT-PCR. PairedRNA samples prepared from psoriatic scales were run in parallelreactions with or without reverse transcriptase (+RT, −RT). PCRreactions were then run using two primer sets (see ExperimentalProcedures of Example 1 hereinbelow) including human keratinocyte cDNAas a positive control. Three lanes contain DNA size markers (100 bpladder), and the bright band in the middle is 500 bp.

[0039]FIG. 5 is a reaction scheme illustrating the reaction mechanism ofthe 12R-lipoxygenase of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

[0040] A recognized feature of psoriasis and other proliferativedermatoses is accumulation in the skin of the unusual arachidonic acidmetabolite, 12R-hydroxyeicosatetraenoic acid (12R-HETE). This hydroxyfatty acid is opposite in chirality to the product of the well known12S-lipoxygenase and heretofore, in mammals, is known only as a productof cytochrome P450s. Mechanistic evidence for a lipoxygenase route to12R-HETE in human psoriatic tissue is provided herein. A12R-lipoxygenase that accounts for the biosynthesis is also described.

[0041] Initially, it is noted that the 12R-lipoxygenase of the presentinvention does not metabolize arachidonic acid directly to 12R-HETE.Rather, the 12R-lipoxygenase of the present invention metabolizesarachidonic acid to 12R-hydroperoxyeicosatetraenoic acid (12R-HPETE),which is then converted to 12R-HETE by commonly found peroxidase enzymesor by non-enzymatic reductants. As would thus be appreciated by onehaving ordinary skill in the art, when the phrase “metabolizesarachidonic acid to 12R-hydroxyeicosatetraenoic acid (12R-HETE)” is usedherein and in the claims, it is meant to refer to and include thetwo-step process described above, i.e. that the 12R-lipoxygenase of thepresent invention metabolizes arachidonic acid to 12R-HPETE, which isthen converted to 12R-HETE by commonly found peroxidase enzymes or bynon-enzymatic reductants.

[0042] Applicants demonstrated retention of the C-12 deuterium ofoctadeuterated arachidonic acid in its conversion to 12R-HETE inincubations of psoriatic scales, indicating the end product is notformed by isomerization from 12S-H(P)ETE via the 12-keto derivative.Secondly, analysis of product formed from [10^(R)-³H]- and[10_(S)-H]-labeled arachidonic acids revealed that 12R-HETE synthesis isassociated with stereospecific removal of the pro-R hydrogen from the10-carbon of arachidonate. This result is compatible with12R-lipoxygenase-catalyzed formation of 12R-HETE and not with aP450-catalyzed route to 12R-HETE in psoriatic scales.

[0043] Applicants then cloned a new lipoxygenase from humankeratinocytes wherein the cDNA and deduced amino acid sequences share≦50% identity to other human lipoxygenases. This enzyme, when expressedin Hela cells, oxygenates arachidonic acid to 12-H(P)ETE, 98% 12R inconfiguration. The 12R-lipoxygenase cDNA is detectable by PCR inpsoriatic scales and as a 2.5 kb mRNA by Northern analysis ofkeratinocytes. Identification of this enzyme extends the knowndistribution of R-lipoxygenases to vertebrates, particularly to mammals,and more particularly to humans, and presents a new target fortherapeutic interventions in psoriasis.

Definitions and Techniques Affecting Gene Products and Genes

[0044] The present invention concerns DNA segments, isolatable fromvertebrate tissue, preferably from mammalian tissue, and more preferablyfrom human tissue, which are free from genomic DNA and which are capableof conferring arachidonic acid metabolism activity in a recombinant hostcell when incorporated into the recombinant host cell. As used herein,the term “mammalian tissue” refers to, among others, normal mammalianepithelial tissues, as exemplified by, but not limited to, humankeratinocytes and to abnormal mammalian epithelial tissues, asexemplified by, but not limited to, psoriatic scales. DNA segmentscapable of conferring arachidonic acid metabolism activity may encodecomplete lipoxygenase polypeptides, cleavage products and biologicallyactively functional domains thereof.

[0045] The terms “lipoxygenase polypeptide”, “lipoxygenase geneproduct”, “lipoxygenase”, “LO”, “12R-LO gene product”, and “12R-LO”, asused in the specification and in the claims refer to proteins havingamino acid sequences which are substantially identical to the respectivenative lipoxygenase amino acid sequences and which are biologicallyactive in that they are capable of reacting with arachidonic acid or arecapable of cross-reacting with an anti-LO antibody raised against alipoxygenase, such as 12R-LO. Such sequences are disclosed herein. Theterms “lipoxygenase polypeptide”, “lipoxygenase gene product”,“lipoxygenase”, “Lox”, “12R-LO gene product”, and “12R-LO” also includeanalogs of lipoxygenase molecules which exhibit at least some biologicalactivity in common with native lipoxygenase, 12R-LO. Furthermore, thoseskilled in the art of mutagenesis will appreciate that other analogs, asyet undisclosed or undiscovered, may be used to construct lipoxygenaseanalogs. There is no need for a “lipoxygenase polypeptide”,“lipoxygenase” or “LO”, or a “12R-LO” to comprise all, or substantiallyall, of the amino acid sequence of the native lipoxygenase genes.Shorter or longer sequences are anticipated to be of use in theinvention.

[0046] The terms “lipoxygenase gene” and “12R-LO gene” refer to any DNAsequence that is substantially identical to a DNA sequence encoding alipoxygenase or 12R-LO as defined above. The terms also refer to RNA, orantisense sequences, compatible with such DNA sequences. A “lipoxygenasegene” or a “12R-LO gene” may also comprise any combination of associatedcontrol sequences.

[0047] The term “substantially identical”, when used to define either alipoxygenase or 12R-LO amino acid sequence, or a lipoxygenase or 12R-LOnucleic acid sequence, means that a particular sequence, for example, amutant sequence, varies from the sequence of a natural lipoxygenase,12R-LO, by one or more deletions, substitutions, or additions, the neteffect of which is to retain at least some of biological activity of thelipoxygenase or the 12R-LO protein. Alternatively, DNA analog sequencesare “substantially identical” to specific DNA sequences disclosed hereinif: (a) the DNA analog sequence is derived from coding regions of thenatural lipoxygenase or 12R-LO gene; or (b) the DNA analog sequence iscapable of hybridization of DNA sequences of (a) under moderatelystringent conditions and which encode biologically active lipoxygenaseor 12R-LO gene; or (c) the DNA sequences are degenerative as a result ofthe genetic code to the DNA analog sequences defined in (a) and/or (b).Substantially identical analog proteins will be greater than about 60%identical to the corresponding sequence of the native protein. Sequenceshaving lesser degrees of similarity but comparable biological activityare considered to be equivalents. In determining nucleic acid sequences,all subject nucleic acid sequences capable of encoding substantiallysimilar amino acid sequences are considered to be substantially similarto a reference nucleic acid sequence, regardless of differences in codonsequences.

Percent Similarity

[0048] Percent similarity may be determined, for example, by comparingsequence information using the GAP computer program, available from theUniversity of Wisconsin Geneticist Computer Group. The GAP programutilizes the alignment method of Needleman et al. 1970, as revised bySmith et al. 1981. Briefly, the GAP program defines similarity as thenumber of aligned symbols (i.e. nucleotides or amino acids) which aresimilar, divided by the total number of symbols in the shorter of thetwo sequences. The preferred default parameters for the GAP programinclude: (1) a unitary comparison matrix (containing a value of 1 foridentities and 0 for non-identities) of nucleotides and the weightedcomparison matrix of Gribskov et al., 1986, as described by Schwartz etal., 1979; (2) a penalty of 3.0 for each gap and an additional 0.01penalty for each symbol and each gap; and (3) no penalty for end gaps.

[0049] The term “homology” describes a mathematically based comparisonof sequence similarities which is used to identify genes or proteinswith similar functions or motifs. Accordingly, the term “homology” issynonymous with the term “similarity” and “percent similarity” asdefined above. Thus, the phrases “substantial homology” or “substantialsimilarity” have similar meanings.

Nucleic Acid Sequences

[0050] In certain embodiments, the invention concerns the use oflipoxygenase genes and gene products, such as the 12R-LO gene product,that include within their respective sequences a sequence which isessentially that of a lipoxygenase or 12R-LO gene, or the correspondingproteins. The term “a sequence essentially as that of lipoxygenase or12R-LO gene or gene product”, means that the sequence substantiallycorresponds to a portion of a lipoxygenase or 12R-LO gene or geneproduct and has relatively few bases or amino acids (whether DNA orprotein) which are not identical to those of a lipoxygenase or 12R-LOgene or gene product, (or a biologically functional equivalent of, whenreferring to proteins). The term “biologically functional equivalent” iswell understood in the art and is further defined in detail herein.Accordingly, sequences which have between about 70% and about 80%; ormore preferably, between about 81% and about 90%; or even morepreferably, between about 91% and about 99%; of amino acids which areidentical or functionally equivalent to the amino acids of alipoxygenase or 12R-LO gene or gene product, will be sequences which are“essentially the same”.

[0051] Lipoxygenase and 12R-LO genes which have functionally equivalentcodons are also covered by the invention. The term “functionallyequivalent codon” is used herein to refer to codons that encode the sameamino acid, such as the six codons for arginine or serine, and also torefer to codons that encode biologically equivalent amino acids (seeTable 1). TABLE 1 Functionally Equivalent Codons. Amino Acids CodonsAlanine Ala A GCA GCC GCG GGU Cysteine Cys C UGC UGU Aspartic Acid Asp DGAC GAU Glumatic acid Glu E GAA GAG Phenylalanine Phe F UUC UUU GlycineGly G GGA GGC GGG GGU Histidine His H CAC CAU Isoleucine Ile I AUA AUCAUU Lysine Lys K AAA AAG Leucine Leu L UUA UUG CUA CUC CUG CUUMethionine Met M AUG Asparagine Asn N AAC AAU Proline Pro P CCA CCC CCGCCU Glutamine Gln Q CAA CAG Arginine Arg R AGA AGG CGA CGC CGG CGUSerine Ser S ACG AGU UCA UCC UCG UCU Threonine Thr T ACA ACC ACG ACUValine Val V GUA GUC GUG GUU Tryptophan Trp W UGG Tyrosine Tyr Y UAC UAU

[0052] It will also be understood that amino acid and nucleic acidsequences may include additional residues, such as additional N- orC-terminal amino acids or 5′ or 3′ sequences, and yet still beessentially as set forth in one of the sequences disclosed herein, solong as the sequence meets the criteria set forth above, including themaintenance of biological protein activity where protein expression isconcerned. The addition of terminal sequences particularly applies tonucleic acid sequences which may, for example, include variousnon-coding sequences flanking either of the 5′ or 3′ portions of thecoding region or may include various internal sequences, i.e., introns,which are known to occur within genes.

[0053] The present invention also encompasses the use of DNA segmentswhich are complementary, or essentially complementary, to the sequencesset forth in the specification. Nucleic acid sequences which are“complementary” are those which are base-pairing according to thestandard Watson-Crick complementarity rules. As used herein, the term“complementary sequences” means nucleic acid sequences which aresubstantially complementary, as may be assessed by the same nucleotidecomparison set forth above, or as defined as being capable ofhybridizing to the nucleic acid segment in question under relativelystringent conditions such as those described herein. A particularexample of a contemplated complementary nucleic acid segment is anantisense oligonucleotide.

[0054] Nucleic acid hybridization will be affected by such conditions assalt concentration, temperature, or organic solvents, in addition to thebase composition, length of the complementary strands, and the number ofnucleotide base mismatches between the hybridizing nucleic acids, aswill be readily appreciated by those skilled in the art. Stringenttemperature conditions will generally include temperatures in excess of30° C., typically in excess of 37° C., and preferably in excess of 45°C. Stringent salt conditions will ordinarily be less than 1,000 mM,typically less than 500 mM, and preferably less than 200 mM. However,the combination of parameters is much more important than the measure ofany single parameter. (See, e.g., Wetmur & Davidson, 1968).

[0055] Probe sequences may also hybridize specifically to duplex DNAunder certain conditions to form triplex or other higher order DNAcomplexes. The preparation of such probes and suitable hybridizationconditions are well known in the art.

[0056] As used herein, the term “DNA segment” refers to a DNA moleculewhich has been isolated free of total genomic DNA of a particularspecies. Furthermore, a DNA segment encoding a lipoxygenase or 12R-LOgene product refers to a DNA segment which contains lipoxygenase or12R-LO coding sequences, yet is isolated away from, or purified freefrom, total genomic DNA of Homo sapiens. Included within the term “DNAsegment” are DNA segments and smaller fragments of such segments, andalso recombinant vectors, including, for example, plasmids, cosmids,phages, viruses, and the like.

[0057] Similarly, a DNA segment comprising an isolated or purifiedlipoxygenase or 12R-LO gene refers to a DNA segment includinglipoxygenase or 12R-LO coding sequences isolated substantially away fromother naturally occurring genes or protein encoding sequences. In thisrespect, the term “gene” is used for simplicity to refer to a functionalprotein, polypeptide or peptide encoding unit. As will be understood bythose in the art, this functional term includes both genomic sequencesand cDNA sequences. “Isolated substantially away from other codingsequences” means that the gene of interest, in this case, thelipoxygenase or 12R-LO gene, forms the significant part of the codingregion of the DNA segment, and that the DNA segment does not containlarge portions of naturally-occurring coding DNA, such as largechromosomal fragments or other functional genes or cDNA coding regions.Of course, this refers to the DNA segment as originally isolated, anddoes not exclude genes or coding regions later added to the segment bythe hand of man.

[0058] In particular embodiments, the invention concerns isolated DNAsegments and recombinant vectors incorporating DNA sequences whichencode a 12R-LO protein that includes within its amino acid sequence theamino acid sequence of SEQ ID NO:2. In other particular embodiments, theinvention concerns isolated DNA segments and recombinant vectorsincorporating DNA sequences which encode a protein that includes withinits amino acid sequence the amino acid sequence of the 12R-LO proteincorresponding to human keratinocytes.

[0059] It will also be understood that this invention is not limited tothe particular nucleic acid and amino acid sequences of SEQ ID NOS:1 and2. Recombinant vectors and isolated DNA segments may therefore variouslyinclude the 12R-LO encoding region itself, include coding regionsbearing selected alterations or modifications in the basic codingregion, or include encoded larger polypeptides which neverthelessinclude 12R-LO encoding regions or may encode biologically functionalequivalent proteins or peptides which have variant amino acid sequences.

[0060] In certain embodiments, the invention concerns isolated DNAsegments and recombinant vectors which encode a protein or peptide thatincludes within its amino acid sequence an amino acid sequenceessentially as set forth in SEQ ID NO:2. Naturally, where the DNAsegment or vector encodes a full length 12R-LO gene product, the mostpreferred sequence is that which is essentially as set forth in SEQ IDNO:1 and which encode a protein that exhibits arachidonic acidreactivity in human keratinocytes, as may be determined by HPLCanalysis, as disclosed herein.

[0061] The term “a sequence essentially as set forth in SEQ ID NO:2”means that the sequence substantially corresponds to a portion of SEQ IDNO:2 and has relatively few amino acids which are not identical to, or abiologically functional equivalent of, the amino acids of SEQ ID NO:2.The term “biologically functional equivalent” is well understood in theart and is further defined in detail herein. Accordingly, sequences,which have between about 70% and about 80%; or more preferably, betweenabout 81% and about 90%; even more preferably, between about 91% andabout 99%; of amino acids which are identical or functionally equivalentto the amino acids of SEQ ID NO:2, will be sequences which are“essentially as set forth in SEQ ID NO:2”.

[0062] In particular embodiments, the invention concerns gene therapymethods that use isolated DNA segments and recombinant vectorsincorporating DNA sequences which encode a protein that includes withinits amino acid sequence an amino acid sequence in accordance with SEQ IDNO:2, SEQ ID NO:2 being derived from keratinocytes from Homo sapiens. Inother particular embodiments, the invention concerns isolated DNAsequences and recombinant DNA vectors incorporating DNA sequences whichencode a protein that includes within its amino acid sequence the aminoacid sequence of the 12R-LO protein from human keratinocytes.

[0063] In certain other embodiments, the invention concerns isolated DNAsegments and recombinant vectors that include within their sequence anucleic acid sequence essentially as set forth in SEQ ID NO:1. The term“essentially as set forth in SEQ ID NO:1” is used in the same sense asdescribed above and means that the nucleic acid sequence substantiallycorresponds to a portion of SEQ ID NO:1, respectively, and hasrelatively few codons which are not identical, or functionallyequivalent, to the codons of SEQ ID NO:1, respectively. Again, DNAsegments which encode gene products exhibiting arachidonic acidmetabolism activity of the 12R-LO gene product will be most preferred.The term “functionally equivalent codon” is used herein to refer tocodons that encode the same amino acid, such as the six codons forarginine or serine, and also to refer to codons that encode biologicallyequivalent amino acids (see Table 1).

[0064] The nucleic acid segments of the present invention, regardless ofthe length of the coding sequence itself, may be combined with other DNAsequences, such as promoters, enhancers, polyadenylation signals,additional restriction enzyme sites, multiple cloning sites, othercoding segments, and the like, such that their overall length may varyconsiderably. It is therefore contemplated that a nucleic acid fragmentof almost any length may be employed, with the total length preferablybeing limited by the ease of preparation and use in the intendedrecombinant DNA protocol. For example, nucleic acid fragments may beprepared which include a short stretch complementary to SEQ ID NO:1,such as about 10 nucleotides, and which are up to 10,000 or 5,000 basepairs in length, with segments of 3,000 being preferred in certaincases. DNA segments with total lengths of about 1,000, 500, 200, 100 andabout 50 base pairs in length are also contemplated to be useful.

[0065] The DNA segments of the present invention encompass biologicallyfunctional equivalent 12R-LO proteins and peptides. Such sequences mayrise as a consequence of codon redundancy and functional equivalencywhich are known to occur naturally within nucleic acid sequences and theproteins thus encoded. Alternatively, functionally equivalent proteinsor peptides may be created via the application of recombinant DNAtechnology, in which changes in the protein structure may be engineered,based on considerations of the properties of the amino acids beingexchanged. Changes designed by man may be introduced through theapplication of site-directed mutagenesis techniques, e.g., to introduceimprovements to the antigenicity of the protein or to test 12R-LOmutants in order to examine arachidonic acid reactivity at the molecularlevel.

[0066] If desired, one may also prepare fusion proteins and peptides,e.g., where the 12R-LO coding region is aligned within the sameexpression unit with other proteins or peptides having desiredfunctions, such as for purification or immunodetection purposes (e.g.,proteins which may be purified by affinity chromatography and enzymelabel coding regions, respectively).

[0067] Recombinant vectors form important further aspects of the presentinvention. Particularly useful vectors are contemplated to be thosevectors in which the coding portion of the DNA segment is positionedunder the control of a promoter. The promoter may be in the form of thepromoter which is naturally associated with the 12R-LO gene, e.g., inkeratinocytes, as may be obtained by isolating the 5′ non-codingsequences located upstream of the coding segment or exon, for example,using recombinant cloning and/or PCR technology, in connection with thecompositions disclosed herein.

[0068] In other embodiments, it is contemplated that certain advantageswill be gained by positioning the coding DNA segment under the controlof a recombinant, or heterologous, promoter. As used herein, arecombinant or heterologous promoter is intended to refer to a promoterthat is not normally associated with a 12R-LO gene in its naturalenvironment. Such promoters may include promoters isolated frombacterial, viral, eukaryotic, or mammalian cells. Naturally, it will beimportant to employ a promoter that effectively directs the expressionof the DNA segment in the cell type chosen for expression. The use ofpromoter and cell type combinations for protein expression is generallyknown to those of skill in the art of molecular biology, for example,see Sambrook et al., 1989, specifically incorporated herein byreference. The promoters employed may be constitutive, or inducible, andcan be used under the appropriate conditions to direct high levelexpression of the introduced DNA segment, such as is advantageous in thelarge-scale production of recombinant proteins or peptides. Appropriatepromoter systems contemplated for use in high-level expression include,but are not limited to, the vaccina virus promoter and the baculoviruspromoter, which are more fully described below.

[0069] As mentioned above, in connection with expression embodiments toprepare recombinant 12R-LO proteins and peptides, it is contemplatedthat longer DNA segments will most often be used, with DNA segmentsencoding the entire 12R-LO protein, functional domains or cleavageproducts thereof, being most preferred. However, it will be appreciatedthat the use of shorter DNA segments to direct the expression of 12R-LOpeptides or epitopic core regions, such as may be used to generateanti-12R-LO antibodies, also falls within the scope of the invention.

[0070] DNA segments which encode peptide antigens from about 15 to about50 amino acids in length, or more preferably, from about 15 to about 30amino acids in length are contemplated to be particularly useful. DNAsegments encoding peptides will generally have a minimum coding lengthin the order of about 45 to about 150, or to about 90 nucleotides. DNAsegments encoding full length proteins may have a minimum coding lengthon the order of about 2,500 nucleotides for a protein in accordance withSEQ ID NO:2.

[0071] Naturally, the present invention also encompasses DNA segmentswhich are complementary, or essentially complementary, to the sequenceset forth in SEQ ID NO:1. The terms “complementary” and “essentiallycomplementary” are defined above. Excepting intronic or flankingregions, and allowing for the degeneracy of the genetic code, sequenceswhich have between about 70% and about 80%; or more preferably, betweenabout 81% and about 90%; or even more preferably, between about 91% andabout 99%; of nucleotides which are identical or functionally equivalent(i.e. encoding the same amino acid) of nucleotides of SEQ ID NO:1, willbe sequences which are “essentially as set forth in SEQ ID NO:1”.Sequences which are essentially the same as those set forth in SEQ IDNO:1 may also be functionally defined as sequences which are capable ofhybridizing to a nucleic acid segment containing the complement of SEQID NO:1 under relatively stringent conditions. Suitable relativelystringent hybridization conditions are described herein and will be wellknown to those of skill in the art.

Biologically Functional Equivalents

[0072] As mentioned above, modification and changes may be made in thestructure of the lipoxygenase proteins and peptides, including 12R-LO,described herein and still obtain a molecule having like or otherwisedesirable characteristics. For example, certain amino acids may besubstituted for other amino acids in a protein structure withoutappreciable loss of interactive capacity with structures such as, forexample, C-10 carbon of arachidonic acid. Since it is the interactivecapacity and nature of a protein that defines that protein's biologicalfunctional activity, certain amino acid sequence substitutions can bemade in a protein sequence (or, of course, its underlying DNA codingsequence) and nevertheless obtain a protein with like or evencountervailing properties (e.g., antagonistic v. agonistic). It is thuscontemplated by the inventors that various changes may be made in thesequence of the lipoxygenase proteins and peptides, including 12R-LO,(or underlying DNA) without appreciable loss of their biological utilityor activity.

[0073] It is also well understood by the skilled artisan that, inherentin the definition of a biologically functional equivalent protein orpeptide, is the concept that there is a limit to the number of changesthat may be made within a defined portion of the molecule and stillresult in a molecule with an acceptable level of equivalent biologicalactivity. Biologically functional equivalent peptides are thus definedherein as those peptides in which certain, not most or all, of the aminoacids may be substituted. Of course, a plurality of distinctproteins/peptides with different substitutions may easily be made andused in accordance with the invention.

[0074] It is also well understood that where certain residues are shownto be particularly important to the biological or structural propertiesof a protein or peptide, e.g., residues in active sites, such residuesmay not generally be exchanged. This is the case in the presentinvention, where if any changes, for example, in an iron binding-moietyof 12R-LO that render the peptide incapable of metabolism of arachidonicacid to 12R-hydroxyeicosatetraenoic acid would result in a loss ofutility of the resulting peptide for the present invention.

[0075] Amino acid substitutions, such as those which might be employedin modifying the lipoxygenase proteins and peptides, including 12R-LO,described herein, are generally based on the relative similarity of theamino acid side-chain substituents, for example, their hydrophobicity,hydrophilicity, charge, size, and the like. An analysis of the size,shape and type of the amino acid side-chain substituents reveals thatarginine, lysine and histidine are all positively charged residues; thatalanine, glycine and serine are all a similar size; and thatphenylalanine, tryptophan and tyrosine all have a generally similarshape. Therefore, based upon these considerations, arginine, lysine andhistidine; alanine, glycine and serine; and phenylalanine, tryptophanand tyrosine; are defined herein as biologically functional equivalents.

[0076] In making such changes, the hydropathic index of amino acids maybe considered. Each amino acid has been assigned a hydropathic index onthe basis of their hydrophobicity and charge characteristics, these are:isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8);cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine(−0.4); threionine (−0.7); serine (−0.8); tryptophan (−0.9); tyrosine(−1.3); proline (−1.6); histidine (−3.2); -glutamate (−3.5); glutamine(−3.5); aspartate (−3.5); asparagine (−3.5); lysine (−3.9); and arginine(−4.5).

[0077] The importance of the hydropathic amino acid index in conferringinteractive biological function on a protein is generally understood inthe art (Kyle & Doolittle, 1982, incorporated herein by reference). Itis known that certain amino acids may be substituted for other aminoacids having a similar hydropathic index or score and still retain asimilar biological activity. In making changes based upon thehydropathic index, the substitution of amino acids whose hydropathicindices are within ±2 is preferred, those which are within ±1 areparticularly preferred, and those within ±0.5 are even more particularlypreferred.

[0078] It is also understood in the art that the substitution of likeamino acids can be made effectively on the basis of hydrophilicity. U.S.Pat. No. 4,554,101, incorporated herein by reference, states that thegreatest local average hydrophilicity of a protein, as governed by thehydrophilicity of its adjacent amino acids, correlates with itsimmunogenicity and antigenicity, i.e. with a biological property of theprotein. It is understood that an amino acid can be substituted foranother having a similar hydrophilicity value and still obtain abiologically equivalent protein.

[0079] As detailed in U.S. Pat. No. 4,554,101, the followinghydrophilicity values have been assigned to amino acid residues:arginine (+3.0); lysine (+3.0); aspartate (+3.0±1); glutamate (+3.0±1);serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0);threonine (−0.4); proline (−0.5±1); alanine (−0.5); histidine (−0.5);cysteine (−1.0); methionine (−1.3); valine (−1.5); leucine (−1.8);isoleucine (−1.8); tyrosine (−2.3); phenylalanine (−2.5); tryptophan(−3.4).

[0080] In making changes based upon similar hydrophilicity values, thesubstitution of amino acids whose hydrophilicity values are within ±2 ispreferred, those which are within +1 are particularly preferred, andthose within ±0.5 are even more particularly preferred.

[0081] While discussion has focused on functionally equivalentpolypeptides arising from amino acid changes, it will be appreciatedthat these changes may be effected by alteration of the encoding DNA,taking into consideration also that the genetic code is degenerate andthat two or more codons may code for the same amino acid.

Sequence Modification Techniques

[0082] Modifications to the lipoxygenase proteins and peptides,including 12R-LO, described herein may be carried out using techniquessuch as site directed mutagenesis. Site-specific mutagenesis is atechnique useful in the preparation of individual peptides, orbiologically functional equivalent proteins or peptides, throughspecific mutagenesis of the underlying DNA. The technique furtherprovides a ready ability to prepare and test sequence variants, forexample, incorporating one or more of the foregoing considerations, byintroducing one or more nucleotide sequence changes into the DNA.Site-specific mutagenesis allows the production of mutants through theuse of specific oligonucleotide sequences which encode the DNA sequenceof the desired mutation, as well as a sufficient number of adjacentnucleotides, to provide a primer sequence of sufficient size andsequence complexity to form a stable duplex on both sides of thedeletion junction being traversed. Typically, a primer of about 17 to 30nucleotides in length is preferred, with about 5 to 10 residues on bothsides of the junction of the sequence being altered.

[0083] In general, the technique of site-specific mutagenesis is wellknown in the art as exemplified by publications (e.g., Adelman et al.,1983). As will be appreciated, the technique typically employs a phagevector which exists in both a single stranded and double stranded form.Typical vectors useful in site-directed mutagenesis include vectors suchas the M13 phage (Messing et al., 1981). These phage are readilycommercially available and their use is generally well known to thoseskilled in the art. Double stranded plasmids are also routinely employedin site directed mutagenesis which eliminates the step of transferringthe gene of interest from a plasmid to a phage.

[0084] In general, site-directed mutagenesis in accordance herewith isperformed by first obtaining a single-stranded vector or melting apartthe two strands of a double stranded vector which includes within itssequence a DNA sequence which encodes, for example, the 12R-LO gene. Anoligonucleotide primer bearing the desired mutated sequence is prepared,generally synthetically, for example by the method of Crea et al.(1978). This primer is then annealed with the single-stranded vector,and subjected to DNA polymerizing enzymes such as E. coli polymerase IKlenow fragment, in order to complete the synthesis of themutation-bearing strand. Thus, a heteroduplex is formed wherein onestrand encodes the original non-mutated sequence and the second strandbears the desired mutation. This heteroduplex vector is then used totransform appropriate cells, such as E. coli cells, and clones areselected which include recombinant vectors bearing the mutated sequencearrangement.

[0085] The preparation of sequence variants of the selected gene usingsite-directed mutagenesis is provided as a means of producingpotentially useful 12R-LO or other arachidonic acid metabolizing speciesand is not meant to be limiting as there are other ways in whichsequence variants of these peptides may be obtained. For example,recombinant vectors encoding the desired genes may be treated withmutagenic agents to obtain sequence variants (see, e.g., a methoddescribed by Eichenlaub, 1979) for the mutagenesis of plasmid DNA usinghydroxylamine.

Other Structural Equivalents

[0086] In addition to the lipoxygenase peptidyl compounds describedherein, the inventors also contemplate that other sterically similarcompounds may be formulated to mimic the key portions of the peptidestructure. Such compounds may be used in the same manner as the peptidesof the invention and hence are also functional equivalents. Thegeneration of a structural functional equivalent may be achieved by thetechniques of modeling and chemical design known to those of skill inthe art. It will be understood that all such sterically similarconstructs fall within the scope of the present invention.

Introduction of Gene Products

[0087] Where the gene itself is employed to introduce the gene products,a convenient method of introduction will be through the use of arecombinant vector which incorporates the desired gene, together withits associated control sequences. The preparation of recombinant vectorsis well known to those of skill in the art and described in manyreferences, such as, for example, Sambrook et al. (1989), specificallyincorporated herein by reference.

[0088] In vectors, it is understood that the DNA coding sequences to beexpressed, in this case those encoding the lipoxygenase gene products,are positioned adjacent to and under the control of a promoter. It isunderstood in the art that to bring a coding sequence under the controlof such a promoter, one generally positions the 5′ end of thetranscription initiation site of the transcriptional reading frame ofthe gene product to be expressed between about 1 and about 50nucleotides “downstream” of (i.e., 3′ of) the chosen promoter. One mayalso desire to incorporate into the transcriptional unit of the vectoran appropriate polyadenylation site (e.g., 5′-AATAAA-3′), if one was notcontained within the original inserted DNA. Typically, these poly Aaddition sites are placed about 30 to 2000 nucleotides “downstream” ofthe coding sequence at a position prior to transcription termination.

[0089] While use of the control sequences of the specific gene (i.e.,the 12R-LO promoter for 12R-LO) will be preferred, there is no reasonwhy other control sequences could not be employed, so long as they arecompatible with the genotype of the cell being treated. Thus, one maymention other useful promoters by way of example, including, e.g., anSV40 early promoter, a long terminal repeat promoter from retrovirus, anactin promoter, a heat shock promoter, a metallothionein promoter, andthe like.

[0090] As is known in the art, a promoter is a region of a DNA moleculetypically within about 100 nucleotide pairs in front of (upstream of)the point at which transcription begins (i.e., a transcription startsite). That region typically contains several types of DNA sequenceelements that are located in similar relative positions in differentgenes. As used herein, the term “promoter” includes what is referred toin the art as an upstream promoter region, a promoter region or apromoter of a generalized eukaryotic RNA Polymerase II transcriptionunit.

[0091] Another type of discrete transcription regulatory sequenceelement is an enhancer. An enhancer provides specificity of time,location and expression level for a particular encoding region (e.g.,gene). A major function of an enhancer is to increase the level oftranscription of a coding sequence in a cell that contains one or moretranscription factors that bind to that enhancer.

[0092] Unlike a promoter, an enhancer can function when located atvariable distances from transcription start sites so long as a promoteris present.

[0093] As used herein, the phrase “enhancer-promoter” means a compositeunit that contains both enhancer and promoter elements. Anenhancer-promoter is operatively linked to a coding sequence thatencodes at least one gene product. As used herein, the phrase“operatively linked” means that an enhancer-promoter is connected to acoding sequence in such a way that the transcription of that codingsequence is controlled and regulated by that enhancer-promoter. Meansfor operatively linking an enhancer-promoter to a coding sequence arewell known in the art. As is also well known in the art, the preciseorientation and location relative to a coding sequence whosetranscription is controlled, is dependent inter alia upon the specificnature of the enhancer-promoter. Thus, a TATA box minimal promoter istypically located from about 25 to about 30 base pairs upstream of atranscription initiation site and an upstream promoter element istypically located from about 100 to about 200 base pairs upstream of atranscription initiation site. In contrast, an enhancer can be locateddownstream from the initiation site and can be at a considerabledistance from that site.

[0094] An enhancer-promoter used in a vector construct of the presentinvention can be any enhancer-promoter that drives expression in a cellto be transfected. By employing an enhancer-promoter with well-knownproperties, the level and pattern of gene product expression can beoptimized.

[0095] For introduction of, for example, the 12R-LO gene, it is proposedthat one will desire to preferably employ a vector construct that willdeliver the desired gene to the affected cells. This will, of course,generally require that the construct be delivered to the targeted cells,for example, keratinocytes. It is proposed that this may be achievedmost preferably by introduction of the desired gene through the use of aviral vector to carry the 12R-LO sequence to efficiently infect thecells. These vectors will preferably be an adenoviral, a retroviral, avaccinia viral vector or adeno-associated virus. These vectors arepreferred because they have been successfully used to deliver desiredsequences to cells and tend to have a high infection efficiency.

[0096] Commonly used viral promoters for expression vectors are derivedfrom polyoma, cytomegalovirus, Adenovirus 2, and Simian Virus 40 (SV40).The early and late promoters of SV40 virus are particularly usefulbecause both are obtained easily from the virus as a fragment which alsocontains the SV40 viral origin of replication. Smaller or larger SV40fragments may also be used, provided there is included the approximately250 bp sequence extending from the Hind III site toward the Bgl I sitelocated in the viral origin of replication. Further, it is alsopossible, and often desirable, to utilize promoter or control sequencesnormally associated with the desired gene sequence, provided suchcontrol sequences are compatible with the host cell systems.

[0097] The origin of replication may be provided either by constructionof the vector to include an exogenous origin, such as may be derivedfrom SV40 or other viral (e.g., Polyoma, Adeno, VSV, BPV) source, or maybe provided by the host cell chromosomal replication mechanism. If thevector is integrated into the host cell chromosome, the latter is oftensufficient.

[0098] Where the 12R-LO gene itself is employed it will be mostconvenient to simply use the wild type 12R-LO gene directly. However, itis contemplated that certain regions of the 12R-LO gene may be employedexclusively without employing the entire wild type 12R-LO gene. It isproposed that it will ultimately be preferable to employ the smallestregion needed to regulate the metabolism of arachidonic acid to 12R-HETEso that one is not introducing unnecessary DNA into cells which receiveeither a 12R-LO gene construct. Techniques well known to those of skillin the art, such as the use of restriction enzymes, will allow for thegeneration of small regions of the 12R-LO gene. The ability of theseregions to regulate the metabolism of arachidonic acid to 12R-HETE caneasily be determined by the assays reported in the Examples. In general,techniques for assessing metabolism of arachidonic acid to 12R-HETE arewell known in the art.

[0099] It is also contemplated to be within the scope of the presentinvention to prepare a transgenic non-human animal which expresses the12R-LO gene of the present invention. Preferably, the preparation of atransgenic animal which overexpresses human 12R-LO in the skin of theanimal to establish a psoriasis-like disorder in the animal iscontemplated to be within the scope of the present invention. Apreferred transgenic animal is a mouse.

[0100] Techniques for the preparation of transgenic animals are known inthe art. Exemplary techniques are described in U.S. Pat. No. 5,489,742(transgenic rats); U.S. Pat. Nos. 4,736,866, 5,550,316, 5,614,396,5,625,125 and 5,648,061 (transgenic mice); U.S. Pat. No. 5,573,933(transgenic pigs); 5,162,215 (transgenic avian species) and U.S. Pat.No. 5,741,957 (transgenic bovine species), the entire contents of eachof which are herein incorporated by reference.

[0101] With respect to an exemplary method for the preparation of atransgenic mouse, cloned recombinant or synthetic DNA sequences or DNAsegments encoding 12R-LO are injected into fertilized mouse eggs. Theinjected eggs are implanted in pseudo pregnant females and are grown toterm to provide transgenic mice whose cells express 12R-LO. The injectedsequences are constructed having promoter sequences connected so as toexpress the desired protein in skin tissues of the transgenic mouse.

[0102] As noted above, a recognized feature of psoriasis and otherproliferative dermatoses is accumulation in the skin of the unusualarachidonic acid metabolite, 12R-HETE. Thus, the inhibition of theaccumulation in the skin of 12R-HETE is desirable in the treatment ofpsoriasis and other proliferative dermatoses. Accordingly, thetransgenic mice provide useful models for studying compounds beingtested for their usefulness in treating psoriasis and otherproliferative dermatoses.

Generation of Antibodies

[0103] In still another embodiment, the present invention provides anantibody immunoreactive with a polypeptide of the present invention.Preferably, an antibody of the invention is a monoclonal antibody. Meansfor preparing and characterizing antibodies are well known in the art(See, e.g., Antibodies A Laboratory Manual, E. Howell and D. Lane, ColdSpring Harbor Laboratory, 1988).

[0104] Briefly, a polyclonal antibody is prepared by immunizing ananimal with an immunogen comprising a polypeptide or polynucleotide ofthe present invention, and collecting antisera from that immunizedanimal. A wide range of animal species can be used for the production ofantisera. Typically an animal used for production of anti-antisera is arabbit, a mouse, a rat, a hamster or a guinea pig. Because of therelatively large blood volume of rabbits, a rabbit is a preferred choicefor production of polyclonal antibodies.

[0105] As is well known in the art, a given polypeptide orpolynucleotide may vary in its immunogenicity. It is often necessarytherefore to couple the immunogen (e.g., a polypeptide orpolynucleotide) of the present invention) with a carrier. Exemplary andpreferred carriers are keyhole limpet hemocyanin (KLH) and bovine serumalbumin (BSA). Other albumins such as ovalbumin, mouse serum albumin orrabbit serum albumin can also be used as carriers.

[0106] Means for conjugating a polypeptide or a polynucleotide to acarrier protein are well known in the art and include glutaraldehyde,m-maleimidobencoyl-N-hydroxysuccinimide ester, carbodiimide andbis-biazotized benzidine.

[0107] As is also well known in the art, immunogencity to a particularimmunogen can be enhanced by the use of non-specific stimulators of theimmune response known as adjuvants. Exemplary and preferred adjuvantsinclude complete Freund's adjuvant, incomplete Freund's adjuvants andaluminum hydroxide adjuvant.

[0108] The amount of immunogen used of the production of polyclonalantibodies varies inter alia, upon the nature of the immunogen as wellas the animal used for immunization. A variety of routes can be used toadminister the immunogen (subcutaneous, intramuscular, intradermal,intravenous and intraperitoneal. The production of polyclonal antibodiesis monitored by sampling blood of the immunized animal at various pointsfollowing immunization. When a desired level of immunogenicity isobtained, the immunized animal can be bled and the serum isolated andstored.

[0109] In another aspect, the present invention contemplates a processof producing an antibody immunoreactive with a lipoxygenase polypeptide,such as 12R-LO, the process comprising the steps of (a) transfectingrecombinant host cells with a polynucleotide that encodes thatpolypeptide; (b) culturing the host cells under conditions sufficientfor expression of the polypeptide; (c) recovering the polypeptide; and(d) preparing antibodies to the polypeptide. Preferably, thelipoxygenase polypeptide is capable of metabolizing arachidonic acid.Even more preferably, the present invention provides antibodies preparedaccording to the process described above.

[0110] A monoclonal antibody of the present invention can be readilyprepared through use of well-known techniques such as those exemplifiedin U.S. Pat. No 4,196,265, herein incorporated by reference. Typically,a technique involves first immunizing a suitable animal with a selectedantigen (e.g., a polypeptide or polynucleotide of the present invention)in a manner sufficient to provide an immune response. Rodents such asmice and rats are preferred animals. Spleen cells from the immunizedanimal are then fused with cells of an immortal myeloma cell. Where theimmunized animal is a mouse, a preferred myeloma cell is a murine NS-1myeloma cell.

[0111] The fused spleen/myeloma cells are cultured in a selective mediumto select fused spleen/myeloma cells from the parental cells. Fusedcells are separated from the mixture of non-fused parental cells, forexample, by the addition of agents that block the de novo synthesis ofnucleotides in the tissue culture media. Exemplary and preferred agentsare aminopterin, methotrexate, and azaserine. Aminopterin andmethotrexate block de novo synthesis of both purines and pyrimidines,whereas azaserine blocks only purine synthesis. Where aminopterin ormethotrexate is used, the media is supplemented with hypoxanthine andthymidine as a source of nucleotides. Where azaserine is used, the mediais supplemented with hypoxanthine.

[0112] This culturing provides a population of hybridomas from whichspecific hybridomas are selected. Typically, selection of hybridomas isperformed by culturing the cells by single-clone dilution in microtiterplates, followed by testing the individual clonal supernatants forreactivity with an antigen-polypeptides. The selected clones can then bepropagated indefinitely to provide the monoclonal antibody.

[0113] By way of specific example, to produce an antibody of the presentinvention, mice are injected intraperitoneally with between about 1-200mg of an antigen comprising a polypeptide of the present invention. Blymphocyte cells are stimulated to grow by injecting the antigen inassociation with an adjuvant such as complete Freund's adjuvant (anon-specific stimulator of the immune response containing killedMycobacterium tuberculosis). At some time (e.g., at least two weeks)after the first injection, mice are boosted by injection with a seconddose of the antigen mixed with incomplete Freund's adjuvant.

[0114] A few weeks after the second injection, mice are tail bled andthe sera titered by immunoprecipitation against radiolabeled antigen.Preferably, the process of boosting and titering is repeated until asuitable titer is achieved. The spleen of the mouse with the highesttiter is removed and the spleen lymphocytes are obtained by homogenizingthe spleen with a syringe. Typically, a spleen from an immunized mousecontains approximately 5×10⁷ to 2×10⁸ lymphocytes.

[0115] Mutant lymphocyte cells known as myeloma cells are obtained fromlaboratory animals in which such cells have been induced to grow by avariety of well-known methods. Myeloma cells lack the salvage pathway ofnucleotide biosynthesis. Because myeloma cells are tumor cells, they canbe propagated indefinitely in tissue culture, and are thus denominatedimmortal. Numerous cultured cell lines of myeloma cells from mice andrats, such as murine NS-1 myeloma cells, have been established.

[0116] Myeloma cells are combined under conditions appropriate to fosterfusion with the normal antibody-producing cells from the spleen of themouse or rat injected with the antigen/polypeptide of the presentinvention. Fusion conditions include, for example, the presence ofpolyethylene glycol. The resulting fused cells are hybridoma cells. Likemyeloma cells, hybridoma cells grow indefinitely in culture.

[0117] Hybridoma cells are separated from unfused myeloma cells byculturing in a selection medium such as HAT media (hypoxanthine,aminopterin, thymidine). Unfused myeloma cells lack the enzymesnecessary to synthesize nucleotides from the salvage pathway becausethey are killed in the presence of aminopterin, methotrexate, orazaserine. Unfused lymphocytes also do not continue to grow in tissueculture. Thus, only cells that have successfully fused (hybridoma cells)can grow in the selection media.

[0118] Each of the surviving hybridoma cells produces a single antibody.These cells are then screened for the production of the specificantibody immunoreactive with an antigen/polypeptide of the presentinvention. Single cell hybridomas are isolated by limiting dilutions ofthe hybridomas. The hybridomas are serially diluted many times and,after the dilutions are allowed to grow, the supernatant is tested forthe presence of the monoclonal antibody. The clones producing thatantibody are then cultured in large amounts to produce an antibody ofthe present invention in convenient quantity.

[0119] By use of a monoclonal antibody of the present invention,specific polypeptides and polynucleotide of the invention can berecognized as antigens, and thus identified. Once identified, thosepolypeptides and polynucleotide can be isolated and purified bytechniques such as antibody-affinity chromatography. Inantibody-affinity chromatography, a monoclonal antibody is bound to asolid substrate and exposed to a solution containing the desiredantigen. The antigen is removed from the solution through animmunospecific reaction with the bound antibody. The polypeptide orpolynucleotide is then easily removed from the substrate and purified.

[0120] Detecting a Polynucleotide or a Polypeptide of the PresentInvention

[0121] Alternatively, the present invention provides a process ofdetecting a polypeptide of the present invention, wherein the processcomprises immunoreacting the polypeptides with antibodies preparedaccording to the process described above to form antibody-polypeptideconjugates, and detecting the conjugates.

[0122] In yet another embodiment, the present invention contemplates aprocess of detecting messenger RNA transcripts that encode a polypeptideof the present invention, wherein the process comprises hybridizing themessenger RNA transcripts with polynucleotide sequences that encode thepolypeptide to form duplexes; and detecting the duplex. Alternatively,the present invention provides a process of detecting DNA molecules thatencode a polypeptide of the present invention, wherein the processcomprises hybridizing DNA molecules with a polynucleotide that encodesthat polypeptide to form duplexes; and detecting the duplexes.

Screening Assays

[0123] In yet another aspect, the present invention contemplates aprocess of screening substances for their ability to affect arachidonicacid metabolism. Utilizing the methods and compositions of the presentinvention, screening assays for the testing of candidate substances canbe derived. A candidate substance is a substance which potentially canpromote or inhibit arachidonic acid metabolism, by binding or otherintramolecular interaction, with a lipoxygenase polypeptide, such as12R-LO, that metabolizes arachidonic acid. As noted above, a recognizedfeature of psoriasis and other proliferative dermatoses is accumulationin the skin of the unusual arachidonic acid metabolite,12R-hydroxyeicosatetraenoic acid (12R-HETE). Thus, a candidate substanceidentified according to the screening assay described herein iscontemplated to have the ability to inhibit accumulation in the skin of12R-HETE, and thus have utility in the treatment of psoriasis and otherproliferative dermatoses.

[0124] An exemplary method of screening candidate substances for theirability to modulate arachidonic acid metabolism comprises the steps of:(a) establishing replicate test and control samples that comprisearachidonic acid and a vertebrate lipoxygenase polypeptide capable ofconverting arachidonic acid to 12R-hydroxyeicosatetraenoic acid; (b)administering a candidate substance to test sample but not the controlsample; (c) measuring 12R-hydroxyeicosatetraenoic acid levels in thetest and the control samples; and (d) determining that the candidatecompound modulates arachidonic acid metabolism if the12R-hydroxyeicosatetraenoic acid level measured for the test sample isgreater or less than the 12R-hydroxyeicosatetraenoic acid level measuredfor the control sample. The replicate test and control samples canfurther comprise a cell that expresses a vertebrate lipoxygenasepolypeptide capable of converting arachidonic acid to12R-hydroxyeicosatetraenoic acid. The present invention alsocontemplates a recombinant cell line suitable for use in the exemplarymethod.

[0125] Thus, a screening assay of the present invention generallyinvolves determining the ability of a candidate substance to affectmetabolism of arachidonic acid in a target cell, such as the screeningof candidate substances to identify those that modulate, i.e. inhibit orpromote, metabolism of arachidonic acid. Target cells can be eithernaturally occurring cells known to contain a polypeptide of the presentinvention or transformed cell produced in accordance with a process oftransformation set forth hereinbefore.

[0126] As is well known in the art, a screening assay provides a cellunder conditions suitable for testing arachidonic acid metabolism. Theseconditions include but are not limited to pH, temperature, tonicity, thepresence of relevant factors involved in arachidonic acid metabolism(e.g., metal ions such as for example Ca⁺⁺, growth factor, interleukins,or colony stimulating factors), and relevant modifications to thepolypeptide such as glycosylation or prenylation. It is contemplatedthat a polypeptide of the present invention can be expressed andutilized in a prokaryotic or eukaryotic cell. The host cell can also befractionated into sub-cellular fractions where the receptor can befound. For example, cells expressing the polypeptide can be fractionatedinto the nuclei, the endoplasmic reticulum, vesicles, or the membranesurfaces of the cell.

[0127] pH is preferably from about a value of 6.0 to a value of about8.0, more preferably from about a value of about 6.8 to a value of about7.8 and, most preferably about 7.4. In a preferred embodiment,temperature is from about 20° C. to about 50° C., more preferably fromabout 30° C. to about 40° C. and, even more preferably about 37° C.Osmolality is preferably from about 5 milliosmols per liter (mosm/L) toabout 400 mosm/l and, more preferably from about 200 milliosmols perliter to about 400 mosm/l and, even more preferably from about 290mosm/L to about 310 mosm/L. The presence of factors can be required forthe proper testing of arachidonic acid metabolism in specific cells.Such factors include, for example, the presence and absence (withdrawal)of growth factor, interleukins, or colony stimulating factors. U.S. Pat.No. 5,645,999 also describes exemplary screening assays, and the entirecontents of U.S. Pat. No. 5,645,999 are herein incorporated byreference.

[0128] In one embodiment, a screening assay is designed to be capable ofdiscriminating candidate substances having selective ability to interactwith one or more of the polypeptides of the present invention but whichpolypeptides are without a substantially overlapping activity withanother of those polypeptides identified herein.

[0129] Many substances which promote or inhibit the activity of theother human lipoxygenases have been identified, and thus representsuitable candidate substances for a screening assay as described above.For example, the 5S-lipoxygenase inhibitor zileuton is commericallyavailable from Abbot Laboratories, Abbot Park, Ill. and is marketed as atreatment for asthma. Additionally, suitable candidate substances maycomprise compositions which inhibit the activity of the known12S-lipoxygenase, such as those described in U.S. Pat. No. 5,326,902issued to Seipp et al. on Jul. 5, 1994, U.S. Pat. No. 5,234,933 issuedto Marnett et al. on Aug. 10, 1993, and PCT Publication WO 93/25521 ofJohnson et al. published Dec. 23, 1993. Additional screening assaytechniques are described in the these references, and hence, the entirecontents of the these references are incorporated herein by reference.Other examples of candidate substances would be apparent to those havingordinary skill in the art.

Therapeutic Methods

[0130] A therapeutic method is contemplated according to the presentinvention. Such a method may comprise promoting or inhibiting 12R-LO ina vertebrate by administering an effective amount of a substance thatinhibits or promotes expression of a 12R-LO-encoding nucleic acidsegment in the vertebrate. Examples of such a substance, include, forexample, an antisense oligonucleotide derived from SEQ ID NO:1.Therapeutic methods utilizing antisense oligonucleotides have beendescribed in the art, for example in U.S. Pat. Nos. 5,627,158 and5,734,033, the contents of each of which are herein incorporated byreference.

[0131] The therapeutic method may also comprise a substance thatinhibits or promotes metabolism of arachidonic acid by inhibiting orpromoting the activity of 12R-LO. Such a substance may be identifiedaccording to the screening assay set forth above. A preferred example ofa vertebrate is a mammal. A preferred example of a mammal is a human.Thus, the method may comprise treating a patient suffering from adisorder associated with the metabolism of arachidonic acid by 12R-LO byadministering to the patient an effective 12R-LO modulating amount of asubstance identified according to the screening assay described above.By the term “modulating”, it is contemplated that the substance caneither promote or inhibit the activity of 12R-LO, depending on thedisorder to be treated.

[0132] As noted above, a recognized feature of psoriasis and otherproliferative dermatoses is accumulation in the skin of the unusualarachidonic acid metabolite, 12R-hydroxyeicosatetraenoic acid(12R-HETE). Thus, the inhibition of the accumulation in the skin of12R-HETE is desirable in the treatment of psoriasis and otherproliferative dermatoses. Accordingly, as a preferred example, thecontemplated therapeutic method comprises treating a patient sufferingfrom psoriasis by administering to the patient an effective 12R-LOinhibiting amount of a substance identified according to the screeningassay described above. Alternatively, the contemplated therapeuticmethod comprises treating a patient suffering from psoriasis byadministering an effective amount of a substance that inhibits orpromotes expression of a 12R-LO-encoding nucleic acid segment in thepatient. Such a substance may comprise, for example, an antisenseoligonucleotide derived from SEQ ID NO:1.

[0133] The 12R-LO modulating substance and the substance that inhibitsor promotes expression of a 12R-LO-encoding nucleic acid segment arethus adapted for administration as a pharmaceutical composition.Formulation and dose preparation techniques have been described in theart, see for example, those described in U.S. Pat. No. 5,326,902 issuedto Seipp et al. on Jul. 5, 1994, U.S. Pat. No. 5,234,933 issued toMarnett et al. on Aug. 10, 1993, and PCT Publication WO 93/25521 ofJohnson et al. published Dec. 23, 1993, the entire contents of each ofwhich are herein incorporated by reference.

[0134] For the purposes described above, the identified substances maynormally be administered systemically or partially, usually by oral orparenteral administration. The doses to be administered are determineddepending upon age, body weight, symptom, the desired therapeuticeffect, the route of administration, and the duration of the treatmentetc. In a human adult, the doses per person per administration aregenerally between 1 mg and 500 mg, by oral administration, up to severaltimes per day, and between 1 mg and 100 mg, by parenteral administrationup to several times per day. Since the doses to be used depend uponvarious conditions, as mentioned above, there may be a case in whichdoses are lower than or greater than the ranges specified above.

[0135] Solid compositions for oral administration include compressedtablets, pills, dispersible powders, capsules, and granules. In suchcompositions, one or more of the active substance(s) is or are, admixedwith at least one inert diluent (lactose, mannitol, glucose,hydroxypropylcellulose, microcrystalline cellulose, starch,polyvinylpyrrolidone, magnesium metasilicate alminate, etc.). Thecompositions may also comprise, as is normal practice, additionalsubstances other than inert diluents: e.g. lubricating agents (magnesiumstearate, etc.), disintegrating agents (cellulose, calcium glycolateetc.), and assisting agent for dissolving (glutamic acid, aspartic acid,etc.) stabilizing agent (lactose etc.). The tablets or pills may, ifdesired, be coated with gastric or enteric material (sugar, gelatin,hydroxypropylcellulose or hydroxypropylmethyl cellulose phthalate,etc.). Capsules include soft ones and hard ones.

[0136] Liquid compositions for oral administration includepharmaceutically-acceptable emulsions, solutions, suspensions, syrupsand elixirs. In such compositions, one or more of the activesubstance(s) is or are admixed with inert diluent(s) commonly used inthe art (purified water, ethanol etc.). Besides inert diluents, suchcompositions may also comprise adjuvants (wetting agents, suspendingagents, etc.), sweetening agents, flavoring agents, perfuming agents andpreserving agents.

[0137] Other compositions for oral administration include spraycompositions which may be prepared by known methods and which compriseone or more of the active substance(s). Spray compositions may compriseadditional substances other than inert diluents: e.g. preserving agents(sodium sulfite, etc.), isotonic buffer (sodium chloride, sodiumcitrate, citric acid, etc.). For preparation of such spray compositions,for example, the method described in U.S. Pat. Nos. 2,868,691 or3,095,355 may be used.

[0138] Injections for parenteral administration include sterile aqueousor non-aqueous solution, suspensions and emulsions. In suchcompositions, one or more of active substance(s) is or are admixed withat least one inert aqueous diluent(s) (distilled water for injection,physiological salt solution etc.) or inert non-aqueous diluent(s)(propylene glycol, polyethylene glycol, olive oil, ethanol, POLYSOLBATE80 (registered trade mark) etc.). Injections may comprise additionalother than inert diluents: e.g. preserving agents, wetting agents,emulsifying agents, dispersing agents, stabilizing agents (lactose,etc.), assisting agents such as for dissolving (glutamic acid, asparticacid, etc.). They may be sterilized, for example, by filtration througha bacteria-retaining filter, by incorporation of sterilizing agents inthe compositions or by irradiation. They also be manufactured in theform of sterile solid compositions, for example, by freeze-drying, andwhich can be dissolved in sterile water or some other sterile diluentsfor injection immediately before use.

[0139] Other compositions for administration include liquids forexternal use, and endermic linaments (ointment, etc.), suppositories andpessaries which comprise one or more of the active substance(s) and maybe prepared by known methods.

Screening Assays for a Polypeptide of the Present Invention

[0140] The present invention provides a process of screening abiological sample for the presence of a lipoxygenase polypeptide, suchas 12R-LO. Preferably, the lipoxygenase polypeptide reacts witharachidonic acid. A biological sample to be screened can be a biologicalfluid such as extracellular or intracellular fluid or a cell or tissueextract or homogenate. A biological sample can also be an isolated cell(e.g., in culture) or a collection of cells such as in a tissue sampleor histology sample. A tissue sample can be suspended in a liquid mediumor fixed onto a solid support such as a microscope slide.

[0141] In accordance with a screening assay process, a biological sampleis exposed to an antibody immunoreactive with the polypeptide whosepresence is being assayed. Typically, exposure is accomplished byforming an admixture in a liquid medium that contains both the antibodyand the candidate polypeptide. Either the antibody or the sample withthe polypeptide can be affixed to a solid support (e.g., a column or amicrotiter plate).

[0142] The biological sample is exposed to the antibody under biologicalreaction conditions and for a period of time sufficient forantibody-polypeptide conjugate formation. Biological reaction conditionsinclude ionic composition and concentration, temperature, pH and thelike.

[0143] Ionic composition and concentration can range from that ofdistilled water to a 2 molal solution of NaCl. Preferably, osmolality isfrom about 100 mosmols/l to about 400 mosmols/l and, more preferablyfrom about 200 mosmols/l to about 300 mosmols/l. Temperature preferablyis from about 4° C. to about 100° C., more preferably from about 15° C.to about 50° C. and, even more preferably from about 25° C. to about 40°C. pH is preferably from about a value of 4.0 to a value of about 9.0,more preferably from about a value of 6.5 to a value of about 8.5 and,even more preferably from about a value of 7.0 to a value of about 7.5.The only limit on biological reaction conditions is that the conditionsselected allow for antibody-polypeptide conjugate formation and that theconditions do not adversely affect either the antibody or thepolypeptide.

[0144] Exposure time will vary inter alia with the biological conditionsused, the concentration of antibody and polypeptide and the nature ofthe sample (e.g., fluid or tissue sample). Means for determiningexposure time are well known to one of ordinary skill in the art.Typically, where the sample is fluid and the concentration ofpolypeptide in that sample is about 10⁻¹⁰M, exposure time is from about10 minutes to about 200 minutes.

[0145] The presence of polypeptide in the sample is detected bydetecting the formation and presence of antibody-polypeptide conjugates.Means for detecting such antibody-antigen (e.g., receptor polypeptide)conjugates or complexes are well known in the art and include suchprocedures as centrifugation, affinity chromatography and the like,binding of a secondary antibody to the antibody-candidate receptorcomplex.

[0146] In one embodiment, detection is accomplished by detecting anindicator affixed to the antibody. Exemplary and well known suchindicators include radioactive labels (e.g., ³²P, ¹²⁵I, ¹⁴C), a secondantibody or an enzyme such as horse radish peroxidase. Means foraffixing indicators to antibodies are well known in the art. Commercialkits are available.

Screening Assay for Anti-Polypeptide Antibody

[0147] In another aspect, the present invention provides a process ofscreening a biological sample for the presence of antibodiesimmunoreactive with a lipoxygenase polypeptide, such as 12R-LO.Preferably the lipoxygenase polypeptide reacts with arachidonic acid. Inaccordance with such a process, a biological sample is exposed to alipoxygenase polypeptide, such as 12R-LO, under biological conditionsand for a period of time sufficient for antibody-polypeptide conjugateformation and the formed conjugates are detected.

Screening Assay for Polynucleotide That Encodes a LipoxygenasePolypeptide of the Present Invention

[0148] A DNA molecule and, particularly a probe molecule, can be usedfor hybridizing as an oligonucleotide probe to a DNA source suspected ofencoding a lipoxygenase polypeptide of the present invention, such as12R-LO. Preferably the lipoxygenase polypeptide reacts with arachidonicacid. The probing is usually accomplished by hybridizing theoligonucleotide to a DNA source suspected of possessing a lipoxygenasegene. In some cases, the probes constitute only a single probe, and inothers, the probes constitute a collection of probes based on a certainamino acid sequence or sequences of the polypeptide and account in theirdiversity for the redundancy inherent in the genetic code.

[0149] A suitable source of DNA for probing in this manner is capable ofexpressing a polypeptide of the present invention and can be a genomiclibrary of a cell line of interest. Alternatively, a source of DNA caninclude total DNA from the cell line of interest. Once the hybridizationprocess of the invention has identified a candidate DNA segment, oneconfirms that a positive clone has been obtained by furtherhybridization, restriction enzyme mapping, sequencing and/or expressionand testing.

[0150] Alternatively, such DNA molecules can be used in a number oftechniques including their use as: (1) diagnostic tools to detect normaland abnormal DNA sequences in DNA derived from patient's cells; (2)means for detecting and isolating other members of the polypeptidefamily and related polypeptides from a DNA library potentiallycontaining such sequences; (3) primers for hybridizing to relatedsequences for the purpose of amplifying those sequences; (4) primers foraltering native lipoxygenase DNA sequences; as well as other techniqueswhich rely on the similarity of the DNA sequences to those of the DNAsegments herein disclosed.

[0151] As set forth above, in certain aspects, DNA sequence informationprovided by the invention allows for the preparation of relatively shortDNA (or RNA) sequences (e.g., probes) that specifically hybridize toencoding sequences of a selected lipoxygenase gene. In these aspects,nucleic acid probes of an appropriate length are prepared based on aconsideration of the encoding sequence for a polypeptide of thisinvention. The ability of such nucleic acid probes to specificallyhybridize to other encoding sequences lend them particular utility in avariety of embodiments. Most importantly, the probes can be used in avariety of assays for detecting the presence of complementary sequencesin a given sample. However, other uses are envisioned, including the useof the sequence information for the preparation of mutant speciesprimers, or primers for use in preparing other genetic constructions.

[0152] To provide certain of the advantages in accordance with theinvention, a preferred nucleic acid sequence employed for hybridizationstudies or assays includes probe sequences that are complementary to atleast a 14 to 40 or so long nucleotide stretch of a nucleic acidsequence of the present invention, such as that shown in SEQ ID NO:1. Asize of at least 14 nucleotides in length helps to ensure that thefragment is of sufficient length to form a duplex molecule that is bothstable and selective. Molecules having complementary sequences overstretches greater than 14 bases in length are generally preferred,though, to increase stability and selectivity of the hybrid, and therebyimprove the quality and degree of specific hybrid molecules obtained.One will generally prefer to design nucleic acid molecules havinggene-complementary stretches of 14 to 20 nucleotides, or even longerwhere desired. Such fragments can be readily prepared by, for example,directly synthesizing the fragment by chemical means, by application ofnucleic acid reproduction technology, such as the PCR technology of U.S.Pat. No. 4,683,202, herein incorporated by reference, or by introducingselected sequences into recombinant vectors for recombinant production.

[0153] Accordingly, a nucleotide sequence of the present invention canbe used for its ability to selectively form duplex molecules withcomplementary stretches of the gene. Depending on the applicationenvisioned, one employs varying conditions of hybridization to achievevarying degrees of selectivity of the probe toward the target sequence.For applications requiring a high degree of selectivity, one typicallyemploys relatively stringent conditions to form the hybrids. Forexample, one selects relatively low salt and/or high temperatureconditions, such as provided by 0.02M-0.15M NaCl at temperatures of 50°C. to 70° C. Such conditions are particularly selective, and toleratelittle, if any, mismatch between the probe and the template or targetstrand.

[0154] Of course, for some applications, for example, where one desiresto prepare mutants employing a mutant primer strand hybridized to anunderlying template or where one seeks to isolate polypeptide codingsequences from related species, functional equivalents, or the like,less stringent hybridization conditions are typically needed to allowformation of the heteroduplex. Under such circumstances, one employsconditions such as 0.15M-0.9M salt, at temperatures ranging from 20° C.to 55° C. Cross-hybridizing species can thereby be readily identified aspositively hybridizing signals with respect to control hybridizations.In any case, it is generally appreciated that conditions can be renderedmore stringent by the addition of increasing amounts of formamide, whichserves to destabilize the hybrid duplex in the same manner as increasedtemperature. Thus, hybridization conditions can be readily manipulated,and thus will generally be a method of choice depending on the desiredresults.

[0155] In certain embodiments, it is advantageous to employ a nucleicacid sequence of the present invention in combination with anappropriate means, such as a label, for determining hybridization. Awide variety of appropriate indicator means are known in the art,including radioactive, enzymatic or other ligands, such asavidin/biotin, which are capable of giving a detectable signal. Inpreferred embodiments, one likely employs an enzyme tag such a urease,alkaline phosphatase or peroxidase, instead of radioactive or otherenvironmentally undesirable reagents. In the case of enzyme tags,calorimetric indicator substrates are known which can be employed toprovide a means visible to the human eye or spectrophotometrically, toidentify specific hybridization with complementary nucleicacid-containing samples.

[0156] In general, it is envisioned that the hybridization probesdescribed herein are useful both as reagents in solution hybridizationas well as in embodiments employing a solid phase. In embodimentsinvolving a solid phase, the sample containing test DNA (or RNA) isadsorbed or otherwise affixed to a selected matrix or surface. Thisfixed, single-stranded nucleic acid is then subjected to specifichybridization with selected probes under desired conditions. Theselected conditions depend inter alia on the particular circumstancesbased on the particular criteria required (depending, for example, onthe G+C contents, type of target nucleic acid, source of nucleic acid,size of hybridization probe, etc.). Following washing of the hybridizedsurface so as to remove nonspecifically bound probe molecules, specifichybridization is detected, or even quantified, by means of the label.

Assay Kits

[0157] In another aspect, the present invention contemplates diagnosticassay kits for detecting the presence of a polypeptide of the presentinvention in biological samples, where the kits comprise a firstcontainer containing a first antibody capable of immunoreacting with thepolypeptide, with the first antibody present in an amount sufficient toperform at least one assay. Preferably, the assay kits of the inventionfurther comprise a second container containing a second antibody thatimmunoreacts with the first antibody. More preferably, the antibodiesused in the assay kits of the present invention are monoclonalantibodies. Even more preferably, the first antibody is affixed to asolid support. More preferably still, the first and second antibodiescomprise an indicator, and, preferably, the indicator is a radioactivelabel or an enzyme.

[0158] The present invention also contemplates a diagnostic kit forscreening agents. Such a kit can contain a polypeptide of the presentinvention. The kit can contain reagents for detecting an interactionbetween an agent and a receptor of the present invention. The providedreagent can be radiolabelled. The kit can contain a known radiolabelledagent capable of binding or interacting with a receptor of the presentinvention.

[0159] In an alternative aspect, the present invention providesdiagnostic assay kits for detecting the presence, in biological samples,of a polynucleotide that encodes a polypeptide of the present invention,the kits comprising a first container that contains a secondpolynucleotide identical or complementary to a segment of at least 10contiguous nucleotide bases of, as a preferred example, SEQ ID NO:1.

[0160] In another embodiment, the present invention contemplatesdiagnostic assay kits for detecting the presence, in a biologicalsample, of antibodies immunoreactive with a polypeptide of the presentinvention, the kits comprising a first container containing alipoxygenase polypeptide, such as 12R-LO, that immunoreacts with theantibodies, with the polypeptide present in an amount sufficient toperform at least one assay. Preferably, the lipoxygenase polypeptidemetabolizes arachidonic acid. The reagents of the kit can be provided asa liquid solution, attached to a solid support or as a dried powder.Preferably, when the reagent is provided in a liquid solution, theliquid solution is an aqueous solution. Preferably, when the reagentprovided is attached to a solid support, the solid support can bechromatograph media or a microscope slide. When the reagent provided isa dry powder, the powder can be reconstituted by the addition of asuitable solvent. The solvent can be provided.

[0161] The following examples have been included to illustrate preferredmodes of the invention. Certain aspects of the following examples aredescribed in terms of techniques and procedures found or contemplated bythe present inventors to work well in the practice of the invention.These examples are exemplified through the use of standard laboratorypractices of the inventors. In light of the present disclosure and thegeneral level of skill in the art, those of skill will appreciate thatthe following examples are intended to be exemplary only and thatnumerous changes, modifications and alterations can be employed withoutdeparting from the spirit and scope of the invention.

EXAMPLE 1

[0162] In this Example mechanistic evidence is presented that iscompatible only with a lipoxygenase pathway to 12R-HETE in humanpsoriatic skin. The cloning and initial characterization of a12R-lipoxygenase from normal human keratinocytes is also described, thusestablishing the existence of R-lipoxygenases beyond the invertebrateworld.

EXPERIMENTAL PROCEDURES

[0163] Materials—[1-¹⁴C]Arachidonic acid was purchased from NEN(Dupont). [5,6,8,9,11,12,14,15-²H_(a)]Arachidonic acid was from a batchprepared as previously described (Taber et al. (1982) Methods Enzymol.86:366-369); ²H_(a) (d8) was the most abundant labeled species (54%),but the sample also contained d7 (34%), and d6 (9%); as shown inResults, the deuterium content of 12R-HETE formed from this arachidonicacid was compared with that of 15-HETE prepared by reaction with soybeanlipoxygenase (Sigma, type V). [10_(R)-³H]- and [10_(S)-³H]arachidonicacids were prepared from methyl 8-ketostearate, a gift from Dr Jin K.Cha (University of Alabama), through the following scheme: (i) reductionwith NaB³H₄, (ii) alkaline ester hydrolysis and preparation of thepentafluorobenzyl (PFB) ester, (iii) tosylation, (iv) resolution of theenantiomers by chiral phase HPLC (Chiralcel OD), (v) displacement of thetosylate with LiAlH₄, (vi) re-oxidation at C-1 with chromic acid, (vii)co-culture of the resulting [⁸R-³H] and [8S-³H]stearic acids mixed with[1-¹⁴C]stearic acid with the fungus Saprolegnia parasilica, and (viii)resolution of the labeled arachidonic acids essentially as has beendescribed previously (Maas et al. (1985) J. Biol. Chem. 260:4217-4228).

[0164] Incubation with Deuterated Arachidonic Acid—A sample of psoriaticscales (20-30 mg) was sonicated in 0.5 ml Medium 199 containing 40 mMHepes, and incubated with 100 μM octadeuterated arachidonic acid for 1hour at 37° C. The sample was extracted using the Bligh and Dyer method(Bligh et al. (1959) Can. J. Biochem. Physiol. 37:911-917) and thedeuterated 12-HETE product was purified by RP-HPLC using a Beckman 5μODS Ultrasphere column and a solvent of MeOH/H₂O/HAc (80/20/0.01,v/v/v). Care was taken to allow for the slightly more polar character(earlier elution) of the labeled product compared to unlabeled 12-HETE.The 12-HETE was further purified by SP-HPLC using an Alltech 5μ Econosilcolumn (25×0.46 cm) and a solvent of hexane:isopropanol:glacial aceticacid (100:2:0.1, v/v/v). It was then converted to the pentafluorobenzylester (PFB) derivative and purified again by SP-HPLC using a solvent ofhexane/isopropanol (100:1, v/v). The resulting sample was analyzed on aChiralcel OD HPLC column (25×0.46 cm) using a solvent ofhexane:isopropanol (100:5, v/v) at a flow rate of 1.1 ml/min with UVdetection at 235 nm, as described in Brash et al. (1990) MethodsEnzymol. 187:187-192.

[0165] GC-MS Analysis—HETE PFB esters were analyzed as thetrimethylsilyl ether derivatives by GC-MS in the negative ion/chemicalionization mode using a Nermag RIO-10C instrument, as described in Blairet al. (1990) Methods Enzymol. 187:13-23. Repetitive spectra wereacquired by scanning over the mass range m/z 390-404, encompassing themajor M-PFB ions at m/z 391 (unlabeled HETE) and m/z 399 (d8 analogue),essentially as described previously in Song et al. (1993) J. Biol. Chem.268:6293-6298.

[0166] Experiments with Stereospecifically Labeled Arachidonic Acids—Thespecific activities of the two 10-³H-labeled arachidonic acids wereapproximately 10,000-20,000 DPM ³H per pg. The pro-S [10⁻³H]arachidonicacid was enriched in tritium by incubation with an 8R-lipoxygenase ofPlexaura homomalla as described in principle previously in Hughes et al.(1991) Biochim. Biophys. Acta 1081:347-354. Thestereospecifically-labeled arachidonic acids were admixed with[¹⁴C]arachidonic acid which served as an internal standard formeasurement of tritium retention. The final ³H/¹⁴C ratios were in therange of 1.1-2.6 in different experiments.

[0167] Incubations were conducted in a volume of 0.2 ml 50 mM Tris pH7.5, 100 mM NaCl, using (30,000 CPM ³H of stereospecifically-labeledarachidonic acids (mixed with [¹⁴C]arachidonic acid) and (20 mg aliquotsof psoriatic scales that were known to metabolize arachidonic acid to12-HETE (patient #1) and 15-HETE+12-HETE (patient #2). The scales weresonicated briefly in the buffer and incubated for 90 min at 37° C. Thesamples were extracted with the Bligh and Dyer procedure (Bligh et al.(1959) Can. J. Biochem. Physiol. 37:911-917), including 1 μgtriphenylphosphine to ensure reduction of any hydroperoxides. Productswere purified by RP-HPLC (Beckman 5μ ODS Ultrasphere, solventMeOH/H₂O/HAc (80/20/0.01, v/v/v), by SP-HPLC of the methyl ester(Alltech 5 p Econosil, hexane/isopropanol (100:1, v/v), and then bychiral phase HPLC (Chiralcel OD, hexane/isopropanol (100:2, v/v)). The12R and 12S enantiomers were well resolved on the chiral column withretention times of 14 and 17.5 min respectively, and =1 min of baselineseparation between the peaks. Fractions of 30 sec were collected acrossthe eluting peaks, evaporated to dryness, mixed with scintillant andeach counted for at least 60 min to define the ³H/¹⁴C ratios of thebaseline and the chromatographic peaks. Recovered 12R-HETE contained150-500 CPM over background in the ¹⁴C channel.

[0168] Preparation of RNA. and cDNA synthesis—Samples of human scalphair roots ((30 mainly anagen follicles) or psoriatic scales (100 mg)were placed in 1 ml TRI Reagent (Molecular Research Center, Inc.) andagitated in a bead beater for 20 seconds using autoclaved 200 micronglass beads. Keratinocyte RNA was prepared using 1.5 ml of TRI Reagentdirectly applied to a 10 cm plate of cultured cells and swirled todissolve the RNA and protein. Total RNA was then extracted according tothe manufacturer's instructions. mRNA was prepared from total RNA usingthe Oligotex mRNA Mini Kit (Qiagen). First strand cDNA was preparedusing an oligo-dT-adapter primer. Preparation of hair follicle cDNA withadaptor primers (Marathon kit, Clontech) was performed as described inBrash et al. (1997) Proc. Natl. Acad:Sci. USA 94:6148-6152.

[0169] PCR cloning—PCR reactions were primed with human hair folliclecDNA, keratinocyte cDNA, and in some experiments with cDNA prepared frompsoriatic scales in a 50 μl reaction mixture of 10 mM Tris, pH 8.3, 50mM KCI, 3 mM MgCl₂ with 0.2 mM of each dNTP and 0.25 μl (1.25 units)AmpliTaq DNA polymerase (Perkin Elmer) in a Perkin Elmer 480thermocycler. After addition of cDNA (1 pi from a 50 μl cDNA synthesis)at 940 (hot start), the PCR was programmed as follows: 94° for2 min, 1cycle; 600 for 1 min, 72° for 1 min, 94° for 1 min, 30 cycles; 720 for10 min, 1 cycle, and then the block temperature was held at 4° C. Theprimers were designed based on EST database entry AA649213 from humantonsillar cells. The upstream primer was5′-C-AAC-TTC-CCA-GCG-TCC-ATG-CGT-AAT-CCA-3′ (SEQ ID NO:3) versus thedownstream primer 5′-TG-GTG-TTr-TGG-TCT-CTG-AGG-TTT-TTG-TGT-T-3′ (SEQ IDNO:4), which corresponds to the 3′ end of the open reading frame withthe downstream primer in the UTR region. A band of 431 bp was produced.

[0170] The 5′ RACE was accomplished using the Marathon cDNA AmplicationKit (Clontech) using 4 μg of total RNA from beard hair follicles,according to methods described in Brash et al. (1997) Proc. Natl.Acad:Sci. USA 94:6148-6152. The gene-specific downstream primers were5′-TGGTGTTTTGGTCTCTGAGGTTTTGTGTT-3′ (SEQ ID NO:5) and5′-TTTTTGCTTGTTTGTTTTGTTTTGTTGAA-3′ (SEQ ID NO:6).

[0171] A full length clone was obtained by PCR using primers purified byHPLC and using a proof-reading mixture of Taq/Pwo DNA polymerases(Expand High Fidelity, Boehringer-Mannheim) as described previously inBrash et al. (1997) Proc. Natl. Acad:Sci. USA 94:6148-6152. The upstreamprimer encoded 5′-TTGGGCCTTCGTGTGGCCCTCCA-3′ (SEQ ID NO:7), part of the5′ UTR about 30 bp upstream of the ATG translation start site. Thedownstream primer encoded the C-terminus of the protein:5′-AGC-GCG-CTC-CTA-AAT-AGA-AAT-GCT-3′ (SEQ ID NO:8). After a hot startat 94° C., the reaction conditions were 94°, 2 min, 1 cycle; 60° for 1min, 72° for 2 min, 96° 15 sec, 30 cycles; 72° 10 min, cycle; hold at 4°C.

[0172] DNA sequencing—PCR products were subcloned into the pCR3.1 vector(Invitrogen) and sequenced by automated sequencing on an ABI Prism 377Genetic analyzer and fluorescence-tagged dye terminator cycle sequencing(Perkin Elmer). Sequence similarities were calculated using the JotunHein algorithm of the Megalign program of Lasergene (DNASTAR Inc., WI).

[0173] Expression of cDNA, HPLC analysis of lipoxygenase metabolism—ThePCR products corresponding to the open reading frame of the cDNA wereligated directly into bidirectional pCR3.1 (Invitrogen), clones with thecorrect orientation were selected by restriction enzyme digest, andthese were then expressed by transient transfection in human Hela cellsas described previously in Jisaka et al. (1997) J. Biol. Chem.272:24410-24416. Initially twelve clones in pCR 3.1 were evaluated (tenexpressed with equivalent activity), and subsequently an additional nineclones were expressed in pBluescript SK (four were active). Followingincubation with substrate (50 or 100 μM [1-¹⁴C]arachidonic acid or[1-¹⁴C]linoleic acid) for 30 min at 37° C. in 50 mM Tris (pH 7.5)containing 150 mM NaCl, 0.1 mM CaCl₂, the products were extracted usingthe Bligh and Dyer procedure (Bligh et al. (1959) Can. J. Biochem.Physiol. 37:911-917) and treated with triphenylphosphine to reduce anyhydroperoxides to HETEs. The extracts were analyzed by reversed-phaseHPLC, normal phase HPLC and chiral phase HPLC, as described in Brash etal. (1990) Methods Enzymol. 187:187-192.

[0174] Northern Analysis—Three nylon membranes containing mRNA fromhuman tissues (Clontech, Palo Alto, Calif.) were probed using a³²P-labeled EcoRI/NcoI 648 bp fragment of the new human lipoxygenaseprepared from the plasmid and labeled by Rediprime random priming(Amersham). After hybridization in ExpressHyb solution (Clontech) at 68°C. for 1 hr, the membranes were washed finally in 0.1×SSC/0.1% SDS at50° C. for 40 min and exposed to film. The same procedure was used forNorthern analysis of human keratinocyte mRNA.

[0175] Detection of the mRNA in Human Psoriatic Scales—RNA was preparedusing Tri Reagent (Molecular Research Center, Inc., Cincinnati, Ohio).Identical aliquots of the RNA samples were used in a cDNA synthesisreaction mixture with and without reverse transcriptase. PCR reactionswere run with human keratinocyte cDNA as template, and also withpsoriatic skin cDNA together with a parallel blank reaction withoutreverse transcriptase as a negative control.

[0176] Two pairs of primers were used, 5′-TGCCTGCTGCACTTTGGACC-3′ (SEQID NO:9) with 5′-TGGTCTTCACATCCGGCAACGT-3′ (SEQ ID NO:10) giving a 852bp product, and 5′-CAACTTCCCAGCGTCCATGCGTAATCCA-3′ (SEQ ID NO:11) with5′-TGGTGTTTTGGTCTCTGAGGTTTTTGTGTT-3′ (SEQ ID NO:12) giving a 431 bpproduct. Both reactions were run using an annealing temperature of 60°in the PCR. RESULTS

[0177] Investigation of a Potential Isomerization of 12S- to12R-HETE—One potential pathway to 12R-HETE is via synthesis of12S-H(P)ETE, followed by oxidation to the 12-keto analogue and reductionback to 12R-HETE. To address the possible existence of this pathway inpsoriatic scales, the retention of deuterium in the biosynthesis of12R-HETE from octadeuterated arachidonic acid was measured. Thissubstrate contains a deuterium label at C-12 which would be lost uponformation of a keto intermediate.

[0178] Following incubation of octadeuterated arachidonic acid withpsoriatic scales, the 12-HETE was isolated, the 12R and 12S enantiomerswere resolved by chiral phase HPLC (FIG. 1A), and the deuterium contentof the 12R-HETE was measured by mass spectrometry (FIG. 1B). For directcomparison with a reaction involving no loss of deuterium, the 12R-HETEspectrum in FIG. 1B is compared to that of labeled 15-HETE prepared fromthe same batch of deuterated arachidonic acid using the soybeanlipoxygenase. The deuterium content of the 12R-HETE and the 15-HETE areindistinguishable (and identical to that of d₈-15-HETE formed in thepsoriatic scales, not shown), indicating no loss of label in theformation of 12R-HETE and thus eliminating keto-hydroxy rearrangementsas a route to 12R-HETE in psoriatic skin.

[0179] Stereospecificity of Hydrogen Abstraction in 12R-HETEBiosynthesis—Conversion of arachidonic acid to 12-HETE requires removalof one of the two methylene hydrogens on the 10-carbon. A cytochromeP450 and 12R-lipoxygenase would show different stereoselectivity in thishydrogen abstraction. Cytochrome P450s tend to exhibit a suprafacialrelationship between hydrogen abstraction and oxygen insertion, i.e. thetwo occur on the same face of the substrate (White et al. (1986) J. Am.Chem. Soc. 108:6024-6031; Oliw et al. (1993) Arch. Biochem. Biophys.300:434-439). By contrast, with lipoxygenases the two occur on oppositefaces (an antarafacial relationship) (e.g., Hawkins et al. (1987) J.Biol. Chem. 262:7629-7634; Hamberg et al. (1967) J. Biol. Chem.242:5329-5335; Egmond et al. (1972) Biochem. Biophys. Res. Commun.48:1055-1060; Hamberg et al. (1980) Biochem. Biophys. Res. Commun.95:1090-1097; Maas et al. (1982) J. Biol. Chem. 257:13525-13519).

[0180] This feature of 12R-HETE synthesis was examined by conductingincubations of psoriatic scales with arachidonic acids containing apro-R or pro-S tritium label on the 10-carbon (with [¹⁴C]arachidonicacid included to standardize the measurements of tritium retention). The12-HETE product from each incubation was purified by HPLC and the 12Rand 12S enantiomers were resolved by chiral phase HPLC. The 12Renantiomer accounted for 80-90% of the 12-HETE product from psoriaticskin. The tritium retention was determined from the ³H/¹⁴C ratio byliquid scintillation counting (Table 2).

[0181] Arachidonic acid with a pro-R10-³H label gave rise to 12R-HETEthat had lost virtually all the tritium (Table 2, first column). This isexactly as predicted for catalysis by a 12R-lipoxygenase (Scheme in FIG.5), and indeed it matches the result obtained with the 12R-lipoxygenaseto be described below (Table 2). Using the arachidonic acid substratewith the pro-S ³H label at C-10, the 12R-HETE product retained about 85%of the tritium. This is compatible with results obtained in otherlipoxygenase-catalyzed reactions in which a secondary isotope effectslightly slows the rate of reaction of the ³H-labeled moleculesresulting in less than 100% ³H retention in the product (Maas et al.(1982) J. Biol. Chem. 257:13525-13519; Brash et al. (1986) Biochim.Biophys. Acta 875:256-261). These results indicate no significant P450involvement in 12R-HETE synthesis under the conditions of theseexperiments and directly indicate a 12R-lipoxygenase pathway.

[0182] Molecular Cloning of a Novel Human Lipoxygenase—The initial cloneof a novel human lipoxygenase was obtained using hair follicle andkeratinocyte cDNAs as template and primers based on sequence from ahuman EST (GenBank, AA649213). The published sequence comprisedapproximately 500 bp encoding the 3′ end of the open reading frame and150 bp of 3′ UTR. The sequence clearly encoded a previously undescribedlipoxygenase. The 5′ end of the lipoxygenase transcript was obtained by5′ RACE using human hair follicle cDNA as template. The cDNA encodingthe complete open reading frame (the open reading frame is fromnucleotides 260-2362 inclusive) was then prepared by PCR and subclonedinto the pCR 3.1 vector. Two of the active clones described below weresequenced (FIG. 2).

[0183] The novel lipoxygenase cDNA has approximately 50% similarity insequence to the second type of human 15S-lipoxygenase (Brash et al.(1997) Proc. Natl. Acad Sci. USA 94:6148-6152), and 40% to the human5S-lipoxygenase. It is more distantly related to the 12S- andreticulocyte-type of 15S-lipoxygenase (38% and 35% similarity,respectively). The new human sequence is closely related to a recentlyreported mouse lipoxygenase cDNA ((86% identity) (Krieg et al. (1998)Biochim. Biophys. Acta 1391:7-12).

[0184] Expression of the cDNA—The cDNA was transfected into vacciniainfected Hela cells and after 20 hours the cell sonicates were evaluatedfor lipoxygenase activity by incubation with [¹⁴C]arachidonic acid andHPLC analysis (Experimental Procedures). Reversed-phase HPLC analysiswith on-line recording of UV spectra and radioactive monitoring showed asingle major product with a conjugated diene UV spectrum and whichco-chromatographed with 12-HETE and 8-HETE. The HETEs were collected asa group from RP-HPLC and further analyzed by normal-phase HPLC as shownin FIG. 3A. The single main product was identified as 12-HETE on thebasis of its co-chromatography with the authentic standard and itsidentical UV spectrum (λ_(max) 237 nm, indicative of the 8cis-10transconjugation). Minor amounts of 15-HETE and 11-HETE were present. The12-HETE product was 98% of the 12R configuration (FIG. 3B). The primary12-lipoxygenase product, 12R-HPETE, was detectable in incubations ofbaculovirus/insect cell-expressed enzyme, confirming that the new enzymeis a 12R-lipoxygenase. Linoleic acid was a relatively poor substrate forthe 12R-lipoxygenase compared to arachidonic acid.

[0185] Tissue Expression of the 12R-lipoxygenase—Northern analysis ofhuman keratinocytes using a 12R-lipoxygenase-specific probe gave asingle band of 2.5 kB (FIG. 4A), compatible with the predicted size ofthe mRNA comprising 260 bp of 5′ UTR, 2103 bp open reading frame, and150 bp 3′ UTR. No distinct hybridization was observed by Northernanalysis of three human multiple tissue Northern blots comprising thefollowing tissues from normal subjects: spleen, thymus, prostate,testis, ovary, small intestine, colon, peripheral blood leukocytes,heart, brain, placenta, lung, liver, skeletal muscle, kidney, pancreas,stomach, thyroid, spinal cord, lymph node, trachea, adrenal gland, andbone marrow. The mRNA could be detected by RT-PCR in cDNA prepared fromhuman hair follicles, human foreskin keratinocytes, and (with uncertainprospects for recovery of mRNA) in one of two samples prepared from thescaly discarded skin of subjects with psoriasis (FIG. 4B).

Discussion of Results

[0186] The presence of 12R-HETE in psoriatic lesions is a recognizedfeature of the disease, yet until the isolation of the 12R-lipoxygenasedescribed above, its enzymatic origin has remained elusive. All theknown mammalian lipoxygenases form S configuration hydroperoxides (Funk,C. D. (1993) Prog. Nuc. Acid Res. Mol. Biol. 45:67-98); this biasedagainst a potential 12R-lipoxygenase pathway. Cytochromes P450 canconvert arachidonic acid to a mixture of oxygenated derivatives thatinclude 12R-HETE (Capdevila et al. (1986) Biochem. Biophys. Res. Commun.141:1007-1011; Oliw, E. H. (1993) Biochim. Biophys. Acta 1166:258-263;Bylund et al. (1998) J. Pharmacol. Exp. Ther. 284:51-60), but thedistinctive aspect of 12R-HETE production in human skin is itsappearance together with very few other products (mainly 12S- and15S-HETEs). Direct biochemical characterization of the enzyme proveddifficult using small amounts of human tissue. For example,NADPH-dependence is not definitively diagnostic for a P450-type ofmonooxygenase; lipoxygenases in tissue extracts are sensitive to theredox environment and can show changes in catalytic activity in thepresence of reducing cofactors (Cochran et al. (189) Biochem. Biophys.Res. Commun. 161:1327-1332; Riendeau et al. (1989) Biochem. J.263:65-572; Shornick et al. (1993) J. Biol. Chem. 268:371-376). For allthese reasons, the enzyme responsible for 12R-HETE production waspreviously uncharacterized.

[0187] In the initial series of experiments described herein, amechanistic approach was used to address the enzymatic origin of12R-HETE in psoriatic scales. The first potential route that wasconsidered was a rearrangement from 12S-H(P)ETE through a 12-ketointermediate. Interconversion of 12R- and 12S-HETEs via the ketone isprecedented in rat liver, skin and leukocyte microsomes, although inthese cases the final reduction favors formation of 12S-HETE (Falgueyretet al. (1988) Biochem. Biophys. Res. Commun. 156:1083-1089; Falgueyretet al. (1990) FEBS Lett. 262:197-200). Nonetheless, in principle theformation of 12R-HETE could occur in skin with the known123-lipoxygenase providing the initial substrate. This pathway to12R-HETE was excluded based on the retention of a C-12 deuterium labelduring the biosynthesis (FIG. 1B).

[0188] The other two possibilities, a cytochrome P450 type ofmonooxygenase or a 12R-lipoxygenase, each involve direct oxygenationinto the 12R configuration. In principle, the two pathways can bedistinguished by the initial formation of a 12R-hydroperoxide in thelipoxygenase-catalyzed reaction. This intermediate, however, is readilyreduced in a crude tissue extract and its detection is particularlyproblematic when low levels of the product are formed. An alternativemethod which relies on analysis of 12R-HETE, the common end product ofthe two potential pathways, was adopted. Applicants measured theretention of tritium in the 12R-HETE after incubation of psoriaticscales with arachidonic acid substrates containing a prochiral tritiumlabel on the 10-carbon. Invariably, lipoxygenases catalyze astereoselective oxygenation with removal of the prochiral hydrogen fromthe opposite face of the substrate (Hamberg et al. (1967) J. Biol. Chem.242:5329-5335; Egmond et al. (1972) Biochem. Biophys. Res. Commun.48:1055-1060; Hamberg et al. (1980) Biochem. Biophys. Res. Commun.95:1090-1097; Maas et al. (1982) J. Biol. Chem. 257:13525-13519). Thisis not observed in P450-catalyzed reactions. With P450s, the hydrogenremoval and oxygenation exhibit a suprafacial relationship, often mixedwith an element of stereorandom hydrogen abstraction (White et al.(1986) J. Am. Chem. Soc. 108:6024-6031; Oliw et al. (1993) Arch.Biochem. Biophys. 300:434-439).

[0189] The above described studies in psoriatic scales provided anunequivocal result. The 12R-HETE formed from [10_(R)-³H]arachidonic acidcontained almost no tritium (Table 2). This indicates there is anantarafacial relationship between hydrogen abstraction and 12Roxygenation, a result compatible only with a 12R-lipoxygenase catalyzedtransformation (Scheme in FIG. 5). The human 12R-lipoxygenase that hasbeen cloned and expressed displays the same characteristics (Table 2).

[0190] With the emerging evidence from the mechanism-based experimentsfor the existence of a human 12R-lipoxygenase, a cloning strategysimilar to that had led to discovery of the second type of human15S-lipoxygenase, as described in Brash et al. (1997) Proc. Natl.Acad:Sci. USA 94:6148-6152, was initiated. Applicants also utilizedprimers from a recently released EST sequence (GenBank, AA649213) tobegin characterization of the cDNA of the human 12R-lipoxygenase. TheEST sequence was obtained from human tonsillar cells enriched forgerminal center B cells.

[0191] The 12R-lipoxygenase cDNA is somewhat unusual in having 260 bp of5′ UTR and a short sequence (150 bp) of 3′ UTR. The open reading frameencodes a protein with all the typical characteristics and conservedamino acids of animal lipoxygenases. It also encodes approximately 5 kDof extra sequence, accounted for by an insert of 31 amino acids. Asimilar 31 amino acid sequence, but wherein 6 of 31 amino acids weredifferent, was observed in the recently reported mouse lipoxygenase cDNAreferenced above. (Krieg et al. (1998) Biochim. Biophys. Acta1391:7-12). By reference to the crystal structure of the rabbitreticulocyte 15S-lipoxygenase (Gillmor et al. (1997) Nature Struct.Biol. 4:1003-1009), the extra sequence in the 12R-lipoxygenase islocated after the first alpha-helix of the main C-terminal domain. Inthis position it can be accomodated on the outside of the proteinwithout disruption of the overall tertiary structure. The 31 amino acidinsert includes seven prolines and five arginines. While there is not aperfect consensus sequence of, for example, a proline-rich SH3-bindingdomain (Lepley et al. (1994) J. Biol. Chem. 269:24163-24168), this extrasequence of the 12R-lipoxygenase could well be involved in regulatoryprotein-protein interactions.

[0192] Applicants were able to establish the 12R-lipoxygenase activityof the enzyme expressed in Hela cells (and additionally inbaculovirus/insect cells, not shown), yet the expressed protein has lowcatalytic activity. It expressed with 10-fold lower activity than thereticulocyte-type of 15-lipoxygenase that we used as a positive controlin each experiment. This is similar to observations with the murine8S-lipoxygenase and epidermal-type of 12S-lipoxygenase (Jisaka et al.(1997) J. Biol. Chem. 272:24410-24416; Funk et al. (1996) J. Biol. Chem.271:23338-23344), both of which also express with weak catalyticactivity in vitro. In psoriatic scales, the production of 12R-HETE and15S-HETE are often of the same order of magnitude (Baer et al. (1991) J.Lipid Research 32:341-347). To account for this, either there is a majordifference in the respective levels of the 12R- and 15S-lipoxygenases,or the activity of the 12R-lipoxygenase is increased under naturalcircumstances by protein modification or interactions with othercomponent(s) of the tissue.

[0193] It was established from the previously described cloning of8R-lipoxygenases from coral that the R- and S-lipoxygenases are membersof the same gene family (Brash et al. (1996) J. Biol. Chem.271:20549-20557; Koljak et al. (1997) Science 277:1994-1996).Characterization of the 12R-lipoxygenase now extends the knownoccurrence of R-lipoxygenases beyond the realm of marine and freshwaterinvertebrates. The mRNA for the human 12R-lipoxygenase has been detectedin hair roots, in primary cultures of foreskin keratinocytes, and byPCR, in a sample of psoriatic scales. With the tools made availablethrough molecular cloning the involvement of this enzyme in the cellproliferation and inflammation of psoriasis can be approached accordingto the methods described hereinabove. TABLE 2 Stereospecificity of C-10Hydrooen Abstraction in 12R-HETE Biosynthesis % Tritium Retention in12R-HETE ProR[10-³H]20.4ω6 ProS[10-³H]20.4ω6 Sample substrate substratePsoriatic scales, patient #1 2 85 Psoriatic scales, patient #2 1 8912R-Lipoxygenase^(a) 1 83

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1 12 1 2469 DNA Homo sapiens CDS (260)..(2362) 1 cccagacacc tgctcactcaccaccagctg ggctccgctg ggcctgcccg gcacccaccc 60 cggccaccaa gggcagcagcttttccagaa tttggctggc aggcctagtc accccacctc 120 gccacctcac cactgcacctcggaggccag ccctgtccac tccactctgt gcctggcttc 180 tcttgcctgc cttgggccttcgtgtggccc tccacggtgt ctgggactga gtgcccctct 240 tgcctcctga agagcagcc atggcc acc tac aaa gtc agg gtg gcc aca ggc 292 Met Ala Thr Tyr Lys Val ArgVal Ala Thr Gly 1 5 10 acc gac ctc ttg tcg gga aca cgg gac tcc atc tcactg acc att gtg 340 Thr Asp Leu Leu Ser Gly Thr Arg Asp Ser Ile Ser LeuThr Ile Val 15 20 25 ggg aca caa gga gag agc cat aag cag ctg ctg aac cacttt ggg aga 388 Gly Thr Gln Gly Glu Ser His Lys Gln Leu Leu Asn His PheGly Arg 30 35 40 gac ttt gca act ggg gcg gtg ggc cag tac acc gtg cag tgccct cag 436 Asp Phe Ala Thr Gly Ala Val Gly Gln Tyr Thr Val Gln Cys ProGln 45 50 55 gac ctg ggt gag ctc atc atc atc cgc ctg cac aaa gag cgg tacgcc 484 Asp Leu Gly Glu Leu Ile Ile Ile Arg Leu His Lys Glu Arg Tyr Ala60 65 70 75 ttc ttc ccc aag gac cct tgg tac tgc aac tat gtg cag atc tgtgcc 532 Phe Phe Pro Lys Asp Pro Trp Tyr Cys Asn Tyr Val Gln Ile Cys Ala80 85 90 ccc aac ggc cgt atc tac cac ttc ccc gcc tac cag tgg atg gat ggc580 Pro Asn Gly Arg Ile Tyr His Phe Pro Ala Tyr Gln Trp Met Asp Gly 95100 105 tac gag acc ctg gca ctc cgg gag gcc aca gga aag aca aca gca gat628 Tyr Glu Thr Leu Ala Leu Arg Glu Ala Thr Gly Lys Thr Thr Ala Asp 110115 120 gac tcg ctc ccc gtc ctc ctg gag cac aga aaa gag gag atc aga gcc676 Asp Ser Leu Pro Val Leu Leu Glu His Arg Lys Glu Glu Ile Arg Ala 125130 135 aag cag gac ttc tac cac tgg cga gtc ttt ctt cct ggc ctg ccc agc724 Lys Gln Asp Phe Tyr His Trp Arg Val Phe Leu Pro Gly Leu Pro Ser 140145 150 155 tat gtg cac att ccc agt tac cgc cct ccg gtg cgg agg cat cgcaac 772 Tyr Val His Ile Pro Ser Tyr Arg Pro Pro Val Arg Arg His Arg Asn160 165 170 ccc aac cgg cct gag tgg aat ggc tat att ccg gga ttc cca attctc 820 Pro Asn Arg Pro Glu Trp Asn Gly Tyr Ile Pro Gly Phe Pro Ile Leu175 180 185 atc aac ttt aag gcc acc aag ttc ctg aac tta aat ctc cgc tactcc 868 Ile Asn Phe Lys Ala Thr Lys Phe Leu Asn Leu Asn Leu Arg Tyr Ser190 195 200 ttc ctc aag acg gcc tcc ttc ttc gtc cgc ctg ggg ccc atg gcactg 916 Phe Leu Lys Thr Ala Ser Phe Phe Val Arg Leu Gly Pro Met Ala Leu205 210 215 gct ttc aaa gtc cgc ggc ctg ttg gac tgc aaa cat tcg tgg aagagg 964 Ala Phe Lys Val Arg Gly Leu Leu Asp Cys Lys His Ser Trp Lys Arg220 225 230 235 ctg aag gac att agg aaa att ttc cct ggc aag aaa tct gtcgtc tcc 1012 Leu Lys Asp Ile Arg Lys Ile Phe Pro Gly Lys Lys Ser Val ValSer 240 245 250 gag tac gtg gcc gag cac tgg gca gag gac acc ttc ttt gggtac cag 1060 Glu Tyr Val Ala Glu His Trp Ala Glu Asp Thr Phe Phe Gly TyrGln 255 260 265 tac ctc aac ggc gtc aac ccc ggc ctg atc cgc cgc tgc acgcgg atc 1108 Tyr Leu Asn Gly Val Asn Pro Gly Leu Ile Arg Arg Cys Thr ArgIle 270 275 280 cca gac aag ttc ccc gtc aca gac gac atg gtg gct ccg ttcctg ggc 1156 Pro Asp Lys Phe Pro Val Thr Asp Asp Met Val Ala Pro Phe LeuGly 285 290 295 gag gga acg tgc ttg caa gcg gag ctg gag aag ggg aac atttac ctg 1204 Glu Gly Thr Cys Leu Gln Ala Glu Leu Glu Lys Gly Asn Ile TyrLeu 300 305 310 315 gcc gac tac cgc atc atg gag ggc atc ccc acc gtg gagctc agc ggc 1252 Ala Asp Tyr Arg Ile Met Glu Gly Ile Pro Thr Val Glu LeuSer Gly 320 325 330 cgg aag cag cac cac tgc gcc ccc ctc tgc ctg ctg cacttt gga ccc 1300 Arg Lys Gln His His Cys Ala Pro Leu Cys Leu Leu His PheGly Pro 335 340 345 gag ggc aag atg atg ccc atc gcc atc cag ctc agc cagacc cct ggg 1348 Glu Gly Lys Met Met Pro Ile Ala Ile Gln Leu Ser Gln ThrPro Gly 350 355 360 cca gat tgc ccc atc ttc ctg ccc agt gat tct gag tgggac tgg ctg 1396 Pro Asp Cys Pro Ile Phe Leu Pro Ser Asp Ser Glu Trp AspTrp Leu 365 370 375 cta gcc aag acg tgg gta cgc tat gcg gag ttc tac agccac gag gcc 1444 Leu Ala Lys Thr Trp Val Arg Tyr Ala Glu Phe Tyr Ser HisGlu Ala 380 385 390 395 atc gcc cac ctg ctg gag aca cac ctc att gct gaggcc ttc tgc ctg 1492 Ile Ala His Leu Leu Glu Thr His Leu Ile Ala Glu AlaPhe Cys Leu 400 405 410 gcc ttg ctg agg aac ctg ccc atg tgc cac ccc ctctac aag ctc ctc 1540 Ala Leu Leu Arg Asn Leu Pro Met Cys His Pro Leu TyrLys Leu Leu 415 420 425 atc ccc cat acc cga tac acc gtc cag atc aac agcatt ggc cgg gcc 1588 Ile Pro His Thr Arg Tyr Thr Val Gln Ile Asn Ser IleGly Arg Ala 430 435 440 gtt ctc ctc aat gag ggg ggg ctc tct gcc aag ggcatg tcc ctg ggc 1636 Val Leu Leu Asn Glu Gly Gly Leu Ser Ala Lys Gly MetSer Leu Gly 445 450 455 gtg gaa ggc ttt gct ggg gtg atg gta cgg gct ctgtcg gag ctc acc 1684 Val Glu Gly Phe Ala Gly Val Met Val Arg Ala Leu SerGlu Leu Thr 460 465 470 475 tat gac agc ctc tac ctc ccc aat gac ttt gtggag cgt ggg gtc cag 1732 Tyr Asp Ser Leu Tyr Leu Pro Asn Asp Phe Val GluArg Gly Val Gln 480 485 490 gac ctg cct gga tat tac tac cgc gat gac agcttg gcg gtg tgg aat 1780 Asp Leu Pro Gly Tyr Tyr Tyr Arg Asp Asp Ser LeuAla Val Trp Asn 495 500 505 gca ctg gag aag tat gtg acg gag atc atc acctat tat tac ccg agt 1828 Ala Leu Glu Lys Tyr Val Thr Glu Ile Ile Thr TyrTyr Tyr Pro Ser 510 515 520 gac gca gcc gtg gag ggt gat ccg gaa ttg cagtct tgg gtg cag gaa 1876 Asp Ala Ala Val Glu Gly Asp Pro Glu Leu Gln SerTrp Val Gln Glu 525 530 535 ata ttt aaa gag tgc ctc ctg ggg cgg gag agctca ggc ttc cct agg 1924 Ile Phe Lys Glu Cys Leu Leu Gly Arg Glu Ser SerGly Phe Pro Arg 540 545 550 555 tgc ttg cga acc gtg cct gag ctg atc cgatat gtc act ata gtc atc 1972 Cys Leu Arg Thr Val Pro Glu Leu Ile Arg TyrVal Thr Ile Val Ile 560 565 570 tac acc tgc tct gcc aag cac gct gct gtcaac aca ggc cag atg gag 2020 Tyr Thr Cys Ser Ala Lys His Ala Ala Val AsnThr Gly Gln Met Glu 575 580 585 ttc acc gcc tgg atg ccc aac ttc cca gcgtcc atg cgg aat cca ccg 2068 Phe Thr Ala Trp Met Pro Asn Phe Pro Ala SerMet Arg Asn Pro Pro 590 595 600 att cag act aag ggg ctg acc act ctg gagacc ttc atg gac acg ttg 2116 Ile Gln Thr Lys Gly Leu Thr Thr Leu Glu ThrPhe Met Asp Thr Leu 605 610 615 ccg gat gtg aag acc acg tgc atc acg ctgctg gtg ctc tgg acc ctc 2164 Pro Asp Val Lys Thr Thr Cys Ile Thr Leu LeuVal Leu Trp Thr Leu 620 625 630 635 agc cga gag cct gac gac agg cgg cccctg gga cac ttc ccg gac att 2212 Ser Arg Glu Pro Asp Asp Arg Arg Pro LeuGly His Phe Pro Asp Ile 640 645 650 cac ttc gtg gag gag gcc ccg cgg aggagc ata gag gcg ttc cgc cag 2260 His Phe Val Glu Glu Ala Pro Arg Arg SerIle Glu Ala Phe Arg Gln 655 660 665 cgc ctg aac cag atc tca cac gac atccgc cag cgc aac aag tgc ctt 2308 Arg Leu Asn Gln Ile Ser His Asp Ile ArgGln Arg Asn Lys Cys Leu 670 675 680 ccc atc ccc tac tac tac ctg gac ccggtg ctg att gag aac agc att 2356 Pro Ile Pro Tyr Tyr Tyr Leu Asp Pro ValLeu Ile Glu Asn Ser Ile 685 690 695 tct att taggagcgcg cttcccgtctctcctctccc cattctgtgc cctactattt 2412 Ser Ile 700 tcaacaaaac aaaacaaacaagcaaaaaac acaaaaacct cagagaccaa aacacca 2469 2 701 PRT Homo sapiens 2Met Ala Thr Tyr Lys Val Arg Val Ala Thr Gly Thr Asp Leu Leu Ser 1 5 1015 Gly Thr Arg Asp Ser Ile Ser Leu Thr Ile Val Gly Thr Gln Gly Glu 20 2530 Ser His Lys Gln Leu Leu Asn His Phe Gly Arg Asp Phe Ala Thr Gly 35 4045 Ala Val Gly Gln Tyr Thr Val Gln Cys Pro Gln Asp Leu Gly Glu Leu 50 5560 Ile Ile Ile Arg Leu His Lys Glu Arg Tyr Ala Phe Phe Pro Lys Asp 65 7075 80 Pro Trp Tyr Cys Asn Tyr Val Gln Ile Cys Ala Pro Asn Gly Arg Ile 8590 95 Tyr His Phe Pro Ala Tyr Gln Trp Met Asp Gly Tyr Glu Thr Leu Ala100 105 110 Leu Arg Glu Ala Thr Gly Lys Thr Thr Ala Asp Asp Ser Leu ProVal 115 120 125 Leu Leu Glu His Arg Lys Glu Glu Ile Arg Ala Lys Gln AspPhe Tyr 130 135 140 His Trp Arg Val Phe Leu Pro Gly Leu Pro Ser Tyr ValHis Ile Pro 145 150 155 160 Ser Tyr Arg Pro Pro Val Arg Arg His Arg AsnPro Asn Arg Pro Glu 165 170 175 Trp Asn Gly Tyr Ile Pro Gly Phe Pro IleLeu Ile Asn Phe Lys Ala 180 185 190 Thr Lys Phe Leu Asn Leu Asn Leu ArgTyr Ser Phe Leu Lys Thr Ala 195 200 205 Ser Phe Phe Val Arg Leu Gly ProMet Ala Leu Ala Phe Lys Val Arg 210 215 220 Gly Leu Leu Asp Cys Lys HisSer Trp Lys Arg Leu Lys Asp Ile Arg 225 230 235 240 Lys Ile Phe Pro GlyLys Lys Ser Val Val Ser Glu Tyr Val Ala Glu 245 250 255 His Trp Ala GluAsp Thr Phe Phe Gly Tyr Gln Tyr Leu Asn Gly Val 260 265 270 Asn Pro GlyLeu Ile Arg Arg Cys Thr Arg Ile Pro Asp Lys Phe Pro 275 280 285 Val ThrAsp Asp Met Val Ala Pro Phe Leu Gly Glu Gly Thr Cys Leu 290 295 300 GlnAla Glu Leu Glu Lys Gly Asn Ile Tyr Leu Ala Asp Tyr Arg Ile 305 310 315320 Met Glu Gly Ile Pro Thr Val Glu Leu Ser Gly Arg Lys Gln His His 325330 335 Cys Ala Pro Leu Cys Leu Leu His Phe Gly Pro Glu Gly Lys Met Met340 345 350 Pro Ile Ala Ile Gln Leu Ser Gln Thr Pro Gly Pro Asp Cys ProIle 355 360 365 Phe Leu Pro Ser Asp Ser Glu Trp Asp Trp Leu Leu Ala LysThr Trp 370 375 380 Val Arg Tyr Ala Glu Phe Tyr Ser His Glu Ala Ile AlaHis Leu Leu 385 390 395 400 Glu Thr His Leu Ile Ala Glu Ala Phe Cys LeuAla Leu Leu Arg Asn 405 410 415 Leu Pro Met Cys His Pro Leu Tyr Lys LeuLeu Ile Pro His Thr Arg 420 425 430 Tyr Thr Val Gln Ile Asn Ser Ile GlyArg Ala Val Leu Leu Asn Glu 435 440 445 Gly Gly Leu Ser Ala Lys Gly MetSer Leu Gly Val Glu Gly Phe Ala 450 455 460 Gly Val Met Val Arg Ala LeuSer Glu Leu Thr Tyr Asp Ser Leu Tyr 465 470 475 480 Leu Pro Asn Asp PheVal Glu Arg Gly Val Gln Asp Leu Pro Gly Tyr 485 490 495 Tyr Tyr Arg AspAsp Ser Leu Ala Val Trp Asn Ala Leu Glu Lys Tyr 500 505 510 Val Thr GluIle Ile Thr Tyr Tyr Tyr Pro Ser Asp Ala Ala Val Glu 515 520 525 Gly AspPro Glu Leu Gln Ser Trp Val Gln Glu Ile Phe Lys Glu Cys 530 535 540 LeuLeu Gly Arg Glu Ser Ser Gly Phe Pro Arg Cys Leu Arg Thr Val 545 550 555560 Pro Glu Leu Ile Arg Tyr Val Thr Ile Val Ile Tyr Thr Cys Ser Ala 565570 575 Lys His Ala Ala Val Asn Thr Gly Gln Met Glu Phe Thr Ala Trp Met580 585 590 Pro Asn Phe Pro Ala Ser Met Arg Asn Pro Pro Ile Gln Thr LysGly 595 600 605 Leu Thr Thr Leu Glu Thr Phe Met Asp Thr Leu Pro Asp ValLys Thr 610 615 620 Thr Cys Ile Thr Leu Leu Val Leu Trp Thr Leu Ser ArgGlu Pro Asp 625 630 635 640 Asp Arg Arg Pro Leu Gly His Phe Pro Asp IleHis Phe Val Glu Glu 645 650 655 Ala Pro Arg Arg Ser Ile Glu Ala Phe ArgGln Arg Leu Asn Gln Ile 660 665 670 Ser His Asp Ile Arg Gln Arg Asn LysCys Leu Pro Ile Pro Tyr Tyr 675 680 685 Tyr Leu Asp Pro Val Leu Ile GluAsn Ser Ile Ser Ile 690 695 700 3 28 DNA Homo sapiens 3 caacttcccagcgtccatgc gtaatcca 28 4 30 DNA Homo sapiens 4 tggtgttttg gtctctgaggtttttgtgtt 30 5 30 DNA Homo sapiens 5 tggtgttttg gtctctgagg tttttgtgtt30 6 29 DNA Homo sapiens 6 tttttgcttg tttgttttgt tttgttgaa 29 7 23 DNAHomo sapiens 7 ttgggccttc gtgtggccct cca 23 8 24 DNA Homo sapiens 8agcgcgctcc taaatagaaa tgct 24 9 20 DNA Homo sapiens 9 tgcctgctgcactttggacc 20 10 22 DNA Homo sapiens 10 tggtcttcac atccggcaac gt 22 1128 DNA Homo sapiens 11 caacttccca gcgtccatgc gtaatcca 28 12 30 DNA Homosapiens 12 tggtgttttg gtctctgagg tttttgtgtt 30

What is claimed is:
 1. An isolated and purified biologically activevertebrate lipoxygenase polypeptide capable of converting arachidonicacid to 12R-hydroxyeicosatetraenoic acid.
 2. The polypeptide of claim 1,wherein the polypeptide comprises a mammalian 12R-LO.
 3. The polypeptideof claim 2, wherein the polypeptide comprises a human 12R-LO.
 4. Thepolypeptide of claim 3, wherein the 12R-LO comprises the amino acidsequence of SEQ ID NO:2.
 5. The polypeptide of claim 1, modified to bein detectably labeled form.
 6. An isolated and purified antibody capableof specifically binding to the polypeptide of claim
 1. 7. The antibodyof claim 6 which is a monoclonal antibody.
 8. The antibody of claim 6which is a polyclonal antibody.
 9. A hybridoma cell line which producesthe monoclonal antibody of claim
 8. 10. An isolated and purifiedantibody capable of neutralizing the biological activity of thepolypeptide of claim
 1. 11. The antibody of claim 10 which is amonoclonal antibody.
 12. The antibody of claim 10 which is a polyclonalantibody.
 13. A hybridoma cell line which produces the monoclonalantibody of claim
 11. 14. An isolated and purified nucleic acid segmentcomprising an isolated gene encoding a biologically active vertebratelipoxygenase polypeptide capable of converting arachidonic acid to12R-hydroxyeicosatetraenoic acid.
 15. The nucleic acid segment of claim14, wherein the isolated gene encodes a mammalian 12R-LO.
 16. Thenucleic acid segment of claim 15, wherein the isolated gene encodes ahuman 12R-LO.
 17. The nucleic acid segment of claim 14, wherein theisolated gene encodes a 12R-LO comprising the amino acid sequence of SEQID NO:2.
 18. The nucleic acid segment of claim 17, further defined ascomprising the 12R-LO-encoding nucleic acid sequence of SEQ ID NO:1. 19.The nucleic acid segment of claim 14, further defined as a DNA segment.20. The nucleic acid segment of claim 14, wherein the isolated gene ispositioned under the control of a promoter.
 21. The nucleic acid segmentof claim 14, further defined as a recombinant vector which comprises theisolated gene.
 22. The nucleic acid segment of claim 21, wherein thevector is a recombinant expression vector.
 23. The nucleic acid segmentof claim 21, further defined as a nucleic acid fragment of up to 10,000basepairs in length.
 24. The nucleic acid segment of claim 23, furtherdefined as comprising at least a 1000 nucleotide long contiguous stretchof the nucleic acid sequence of SEQ ID NO:1.
 25. A recombinant host cellcomprising the nucleic acid segment of claim
 14. 26. The recombinanthost cell of claim 25, wherein the host cell is a procaryotic cell. 27.The recombinant host cell of claim 25, wherein the host cell is aeukaryotic cell.
 28. A method of preparing a lipoxygenase polypeptide,comprising: transforming a cell with the nucleic acid segment of claim14 to produce a lipoxygenase under conditions suitable for theexpression of said polypeptide.
 29. A process of detecting in a samplean RNA that encodes the lipoxygenase polypeptide encoded by the nucleicacid of claim 14, said process comprising the steps of: (a) contactingsaid sample under hybridizing conditions with the nucleic acid segmentof claim 14 to form a duplex; and (b) detecting the presence of saidduplex.
 30. A process of producing an antibody immunoreactive with alipoxygenase polypeptide, the process comprising steps of: (a)transfecting a recombinant host cell with the a nucleic acid segment ofclaim 14, which encodes a lipoxygenase polypeptide; (b) culturing thehost cell under conditions sufficient for expression of the polypeptide;(c) recovering the polypeptide; and (d) preparing the antibody to thepolypeptide.
 31. The process of claim 30, wherein the polypeptidecomprises SEQ ID NO:2.
 32. The process of claim 30, wherein the nucleicacid segment comprises SEQ ID NO:1.
 33. An antibody produced by theprocess of claim
 30. 34. A process of detecting a lipoxygenasepolypeptide, the process comprising immunoreacting the polypeptide withan antibody prepared according the process of claim 30 to form anantibody-polypeptide conjugate; and detecting the conjugate.
 35. Aprocess of detecting a messenger RNA transcript that encodes alipoxygenase polypeptide, the process comprising the steps ofhybridizing the messenger RNA transcript with the nucleic acid segmentof claim 14 to form a duplex; and detecting the duplex.
 36. A process ofdetecting a DNA molecule that encodes a lipoxygenase polypeptide, theprocess comprising the steps of hybridizing DNA molecules with thenucleic acid segment of claim 14 to form a duplex; and detecting theduplex.
 37. A diagnostic assay kit for detecting the presence of alipoxygenase polypeptide in a biological sample, the kit comprising afirst container containing a first antibody capable of immunoreactingwith a lipoxygenase polypeptide of claim 1, wherein the first antibodyis present in an amount sufficient to perform at least one assay. 38.The assay kit of claim 37, further comprising a second containercontaining a second antibody that immunoreacts with the first antibody.39. The assay kit of claim 38, wherein the first antibody and the secondantibody comprise monoclonal antibodies.
 40. The assay kit of claim 38,wherein the first antibody is affixed to a solid support.
 41. The assaykit of claim 38, wherein the first and second antibodies each comprisean indicator.
 42. The assay kit of claim 41, wherein the indicator is aradioactive label or an enzyme.
 43. A diagnostic assay kit for detectingthe presence, in biological samples, of a lipoxygenase polypeptide, thekit comprising a first container that contains a nucleic acid segmentidentical or complimentary to a segment of at least ten contiguousnucleotide bases of the nucleic acid segment of claim
 14. 44. Adiagnostic assay kit for detecting the presence, in a biological sample,of an antibody immunoreactive with a lipoxygenase polypeptide, the kitcomprising a first container containing a lipoxygenase polypeptide ofclaim 1 that immunoreacts with the antibody, with the polypeptidepresent in an amount sufficient to perform at least one assay.
 45. Amethod of screening candidate substances for their ability to modulatearachidonic acid metabolism, the method comprising the steps of: (a)establishing replicate test and control samples that comprisearachidonic acid and a vertebrate lipoxygenase polypeptide capable ofconverting arachidonic acid to 12R-hydroxyeicosatetraenoic acid; (b)administering a candidate substance to test sample but not the controlsample; (c) measuring 12R-hydroxyeicosatetraenoic acid levels in thetest and the control samples; and (d) determining that the candidatesubstance modulates arachidonic acid metabolism if the12R-hydroxyeicosatetraenoic acid level measured for the test sample isgreater or less than the 12R-hydroxyeicosatetraenoic acid level measuredfor the control sample.
 46. The method of claim 45, wherein thereplicate test and control samples further comprise a cell thatexpresses a vertebrate lipoxygenase polypeptide capable of convertingarachidonic acid to 12R-hydroxyeicosatetraenoic acid.
 47. A recombinantcell line suitable for use in the method of claim
 46. 48. A method ofmodulating vertebrate 12R-lipoxygenase polypeptide activity in avertebrate, the method comprising the step of administering to thevertebrate an effective amount of a substance capable of modulating the12R-lipoxygenase polypeptide activity in the vertebrate, wherebymodulation of the 12R-lipoxygenase polypeptide activity is accomplished.49. The method of claim 48, wherein the step of administering furthercomprises administering an effective amount of a substance thatmodulates expression of a 12R-LO-encoding nucleic acid segment in thevertebrate.
 50. The method of claim 49, wherein the substance thatmodulates expression of a 12R-LO-encoding nucleic acid segment comprisesan antisense oligonulceotide.
 51. The method of claim 48, wherein the12R-lipoxygenase polypeptide activity comprises converting arachidonicacid to 12R-hydroxyeicosatetraenoic acid, and wherein the step ofadministering comprises administering to the vertebrate an effective12R-LO-modulating amount of a substance capable of modulating 12R-LOmetabolism of arachidonic acid to 12R-hydroxyeicosatetraenoic acid. 52.The method of claim 48, wherein the vertebrate is a mammal.
 53. Themethod of claim 52, wherein the mammal is a human.
 54. A method oftreating a patient suffering from a disorder associated with 12R-LOactivity in the patient, the method comprising the step of administeringto the patient an effective amount of a substance capable of modulatingthe 12R-LO activity in the patient, whereby treatment of the disorder isaccomplished.
 55. The method of claim 54, wherein the step ofadministering further comprises administering an effective amount of asubstance that modulates expression of a 12R-LO-encoding nucleic acidsegment in the patient.
 56. The method of claim 55, wherein thesubstance that modulates expression of a 12R-LO-encoding nucleic acidsegment comprises an antisense oligonulceotide.
 57. The method of claim54, wherein the 12R-lipoxygenase polypeptide activity comprisesconverting arachidonic acid to 12R-hydroxyeicosatetraenoic acid, andwherein the step of administering comprises administering to thevertebrate an effective 12R-LO-modulating amount of a substance capableof modulating 12R-LO metabolism of arachidonic acid to12R-hydroxyeicosatetraenoic acid.
 58. The method of claim 54, whereinthe disorder comprises psoriasis.
 59. A transgenic non-human animalhaving incorporated into its genome a nucleic acid segment comprising anisolated gene encoding a biologically active vertebrate lipoxygenasepolypeptide capable of converting arachidonic acid to12R-hydroxyeicosatetraenoic acid, the nucleic acid segment being presentin said genome in a copy number effective to confer expression in theanimal of the lipoxygenase polypeptide.
 60. The trangenic non-humananimal of claim 59, wherein said nucleic acid segment is further definedas a human 12R-LO encoding segment.
 61. The transgenic non-human animalof claim 60, wherein the expression of the lipoxygenase polypeptide isconferred in skin tissue of the animal.